WO2012075111A1 - Utilisation d'anticorps anti-cd40 en thérapie combinée contre des cancers associés aux cellules b - Google Patents

Utilisation d'anticorps anti-cd40 en thérapie combinée contre des cancers associés aux cellules b Download PDF

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WO2012075111A1
WO2012075111A1 PCT/US2011/062584 US2011062584W WO2012075111A1 WO 2012075111 A1 WO2012075111 A1 WO 2012075111A1 US 2011062584 W US2011062584 W US 2011062584W WO 2012075111 A1 WO2012075111 A1 WO 2012075111A1
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antibody
seq
ser
val
leu
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PCT/US2011/062584
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English (en)
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Johan Baeck
Sanela Bilic
Brett Ewald
John Rediske
Nancy Pryer
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Novartis Ag
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/2878—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the NGF-receptor/TNF-receptor superfamily, e.g. CD27, CD30, CD40, CD95
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/395—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum
    • A61K39/39533—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum against materials from animals
    • A61K39/3955—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum against materials from animals against proteinaceous materials, e.g. enzymes, hormones, lymphokines
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A61P35/02—Antineoplastic agents specific for leukemia
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/2887—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against CD20
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/505—Medicinal preparations containing antigens or antibodies comprising antibodies
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/55—Medicinal preparations containing antigens or antibodies characterised by the host/recipient, e.g. newborn with maternal antibodies

Definitions

  • This invention relates to new uses of anti-CD40 antibodies in the treatment of diseases or conditions associated with neoplastic B-cell growth.
  • the invention is particularly useful for the treatment of patients having CD40 + B-cell lymphomas that are refractory to treatment with the chimeric anti-CD20 monoclonal antibody rituximab and who have been exposed to at least one prior chemotherapeutic regimen.
  • CD40 is a 50-55kDa cell-surface antigen present on the surface of both normal and neoplastic human B-cells. Malignant B-cells from tumors of B-cell lineage express CD40 and appear to depend on CD40 signaling for survival and proliferation.
  • Transformed cells from patients with low- and high-grade B-cell lymphomas, B-cell acute lymphoblastic leukemia, multiple myeloma, chronic lymphocytic leukemia, and Hodgkin's disease express CD40.
  • CD40 expression is also detected in acute myeloblastic leukemia and 50% of AIDS-related lymphomas.
  • Anti-CD40 antibodies and uses thereof have been disclosed, e.g., in co-owned international patent applications published as WO 2005/044294, WO 2005/044304, WO 2005/044305, WO 2005/044306, WO 2005/044307, WO 2005/044854,
  • WO 2007/053661 and WO 2007/053767.
  • CHIR-12.12 human IgGi anti-CD40 monoclonal antibody
  • HCD122 human IgGi anti-CD40 monoclonal antibody
  • any one therapeutic agent may provide a benefit to the patient, further methods are needed to reduce toxicity and to improve treatment outcomes.
  • diseases or conditions can often become refractory to treatment with single-agent therapy, either as a result of initial resistance or resistance that develops during therapy.
  • compositions, uses, and kits useful in the treatment of diseases or conditions associated with neoplastic B-cell growth are provided.
  • the methods, compositions, uses, and kits are directed to combination therapy with bendamustine and an anti-CD40 antibody, wherein at least one therapeutically effective dose of of each of these anti-cancer agents is administered to a patient having a disease or condition that is associated with neoplastic B-cell growth.
  • the invention is particularly useful for the treatment of patients who have previously been administered (i) the chimeric anti-CD20 monoclonal antibody rituximab, or (ii) combination therapy with rituximab and a chemotherapeutic regimen.
  • neoplastic B cells include, but are not limited to, neoplastic B cells derived from lymphomas including low-, intermediate-, and high-grade B cell lymphomas, immunoblastic lymphomas, non- Hodgkin's lymphomas, Hodgkin's disease, Epstein-Barr Virus (EBV) induced lymphomas, and AIDS-related lymphomas, as well as B cell acute lymphoblastic leukemias, myelomas, chronic lymphocytic leukemias, acute myeloblastic leukemias, and the like.
  • neoplastic B cells include, but are not limited to, neoplastic B cells derived from lymphomas including low-, intermediate-, and high-grade B cell lymphomas, immunoblastic lymphomas, non- Hodgkin's lymphomas, Hodgkin's disease, Epstein-Barr Virus (EBV) induced lymphomas, and AIDS-related lymphomas, as well as B cell acute lymphoblast
  • the methods involve combination therapy with (i) an anti-CD40 antibody and (ii) the alkylating agent bendamustine (marketed under the name Treanda®; Cephalon, Inc., Frazer, PA).
  • Combination therapy with an anti-CD40 antibody, such as HCD122, and bendamustine provides a therapeutic benefit that is greater than that provided by the use of either of these anti-cancer agents alone.
  • the combined effect of these two therapies can be greater than the sum of the individual effects of each therapy, i.e., the combination of an anti-CD40 antibody with bendamustine can provide a synergistic therapeutic effect.
  • these two types of anti-cancer agents are used in combination to treat tumors that are refractory to treatment with an anti-CD20 antibody therapy, particularly the anti-CD20 antibody rituximab, where the refractory status of the tumor is a result of initial resistance to the anti-CD20 antibody therapy, or is a result of resistance that develops during one or more time courses of therapy with the anti-CD20 antibody therapy.
  • combination therapy with an anti-CD40 antibody and bendamustine has a synergistic therapeutic effect against tumors that are refractory or non-refractory (i.e., responsive) to anti-CD20 antibody therapy.
  • Administering these two known therapies in combination results in a greater response rate with a more durable response in this patient population than when either agent is administered alone.
  • an anti-CD40 antibody such as HCD122
  • bendamustine will beneficially provide for a greater response rate in patients having a CD40 + B-cell malignancy that is refractory to anti- CD20 antibody therapy, particularly rituximab therapy.
  • the invention thus provides a method for treating a human patient for a disease or condition associated with neoplastic B-cell growth, said method comprising
  • the anti-CD40 antibody (herein “the antibody therapy”) and the bendamustine (herein “the bendamustine therapy”) are administered to the patient at the same time.
  • the antibody therapy may be administered to the patient at exactly the same time as the bendamustine therapy (i.e., the two therapies are administered simultaneously).
  • the antibody therapy may be administered to the patient at approximately the same time as the bendamustine therapy (i.e., the two therapies are not administered at precisely the same time), e.g., during the same visit to a physician or other healthcare professional.
  • the antibody therapy and the bendamustine therapy are not administered to the patient at the same time, but are administered sequentially
  • the methods of the invention may comprise administering a first cycle of bendamustine therapy to the patient before a first dose of the anti-CD40 antibody is administered to the patient.
  • the methods may comprise administering a first cycle of the bendamustine therapy to the patient after a first dose of the anti-CD40 antibody is administered to the patient.
  • the therapies may be administered in such a way that both therapies exert a therapeutic effect on the patient at the same time (i.e., the periods in which each therapy is effective may overlap) although this is not essential.
  • the invention therefore provides a method for treating a human patient for a disease or condition associated with neoplastic B-cell growth, said method comprising administering to the patient one or more doses of an anti-CD40 antibody before, during, or after administering one or more doses of bendamustine.
  • a first cycle of the bendamustine therapy is administered to the patient before a first dose of the anti-CD40 antibody.
  • a first cycle of the bendamustine therapy may be administered from about one (1) day to about two weeks (about 14 days), from about 1 day to about 13 days, from about 1 day to about 12 days, from about 1 day to about 11 days, from about 1 day to about 10 days, from about 1 day to about 9 days, from about 1 day to about 8 days, from about 1 day to about one week (about 7 days), from about 1 day to about 6 days, from about 1 day to about 5 days, from about 1 day to about 4 days, from about 1 day to about 3 days, or from about 1 day to about 2 days, or about 1 day before the first dose of an anti-CD40 antibody is administered to the patient.
  • the bendamustine therapy may be administered about 14 days, about 13 days, about 12 days, about 1 1 days, about 10 days, about 9 days, about 8 days, about 7 days, about 6 days, about 5 days, about 4 days, about 3 days, about 2 days, or about 1 day before a first dose of the anti-CD40 antibody is adminstered to the patient.
  • the antibody therapy may be administered from about one (1) day to about two weeks (about 14 days), from about 1 day to about 13 days, from about 1 day to about 1 1 days, from about 1 day to about 10 days, from about 1 day to about 9 days, from about 1 day to about 8 days, from about 1 day to about one week (about 7 days), from about 1 day to about 6 days, from about 1 day to about 5 days, from about 1 day to about 4 days, from about 1 day to about 3 days, from about 1 day to about 2 days, or about 1 day after the first cycle of bendamustine therapy.
  • the antibody therapy may be administered about 1 day, about 2 days, about 3 days, about 4 days, about 5 days, about 6 days, about 7 days, about 8 days, about 9 days, about 10 days, about 1 1 days, about 12 days, about 13 days, or about 14 days after the first cycle of bendamustine therapy.
  • a first cycle of the bendamustine therapy is administered to the patient after a first dose of the anti-CD40 antibody.
  • a first cycle of bendamustine therapy may be administered about one (1) day to about two weeks (about 14 days), from about 1 day to about 13 days, from about 1 day to about 12 days, from about 1 day to about 1 1 days, from about 1 day to about 10 days, from about 1 day to about 9 days, from about 1 day to about 8 days, from about 1 day to about one week (about 7 days), from about 1 day to about 6 days, from about 1 day to about 5 days, from about 1 day to about 4 days, from about 1 day to about 3 days, or from about 1 day to about 2 days, or about 1 day after the first dose of an anti-CD40 antibody is administered to the patient.
  • a first cycle of bendamustine therapy may be administered about 14 days, about 13 days, about 12 days, about 11 days, about 10 days, about 9 days, about 8 days, about 7 days, about 6 days, about 5 days, about 4 days, about 3 days, about 2 days, or about 1 day after a first dose of the anti-CD40 antibody is adminstered to the patient.
  • the antibody therapy may be administered from about one (1) day to about two weeks (about 14 days), from about 1 day to about 13 days, from about 1 day to about 1 1 days, from about 1 day to about 10 days, from about 1 day to about 9 days, from about 1 day to about 8 days, from about 1 day to about one week (about 7 days), from about 1 day to about 6 days, from about 1 day to about 5 days, from about 1 day to about 4 days, from about 1 day to about 3 days, from about 1 day to about 2 days, or about 1 day before the first cycle of bendamustine therapy.
  • the antibody therapy may be administered about 1 day, about 2 days, about 3 days, about 4 days, about 5 days, about 6 days, about 7 days, about 8 days, about 9 days, about 10 days, about 1 1 days, about 12 days, about 13 days, or about 14 days before the first cycle of bendamustine therapy.
  • the antibody therapy and the bendamustine therapy are administered to a patient in need of treatment for a disease or condition associated with neoplastic B-cell growth, where the administration occurs according to a dosing regimen that comprises multiple cycles of combination therapy with these two anti-cancer agents.
  • the combination therapy comprises administration of these two agents to the patient over multiple cycles (for example, two or more cycles), each of which consists of about 21 days (3 weeks) to about 35 days (about 5 weeks), including, for example, about 28 days (4 weeks).
  • At least one therapeutically effective dose of bendamustine and at least one therapeutically effective dose of the anti-CD40 antibody are administered to the patient by the standard acceptable administration routes for these two anti-cancer agents. Suitable therapeutically effective doses of these two anti-cancer agents are described herein below. In particular embodiments, these two agents are administered intravenously (iv), preferably by infusion over the time periods described elsewhere herein.
  • a patient may receive a maintenance cycle of anti-CD40 antibody therapy.
  • maintenance cycle of anfi-CD40 antibody therapy is intended a time period following combination anti-CD40 antibody/bendamustine therapy during which a patient is administered the anti-CD40 antibody therapy in the absence of bendamustine therapy.
  • the anti-CD40 antibody is administered according to a desired dosing regimen.
  • the anti- CD40 antibody is administered at the same therapeutically effective dose and frequency (for example, bi-weekly) that the antibody was administered during the final cycle of combination anti-CD40 antibody/bendamustine therapy.
  • the length of this maintenance cycle is generally about two weeks to about three months, about two weeks to about two months, about two weeks to about one month, although it can be longer where the health of the patient, history of disease progression, and responsiveness of the patient to follow- up anti-CD40 antibody therapy provide for continued administration of this anti-cancer agent.
  • the invention thus encompasses multiple cycles of combination therapy with an anti-CD40 antibody and bendamustine, optionally followed by a maintenance cycle of anti-CD40 antibody therapy, where the patient undergoing treatment is administered one or more therapeutically effective doses of the antibody and bendamustine according to a suitable dosing regimen.
  • multiple cycles includes, without limitation, two, three, four, five, six, seven, eight, nine, ten or more cycles of combination therapy.
  • the dosing regimen followed within any given cycle of combination therapy may be different, or the same as, the dosing regimen of a prior cycle, or a subsequent cycle, of combination therapy, so long as the patient is administered at least one therapeutically effective dose of bendamustine and at least one therapeutically effective dose of the anti-CD40 antibody during a given cycle of combination therapy with these two anti-cancer agents.
  • multiple cycles of combination therapy with these two agents may be given successively, i.e., without interruption or time period off of combination therapy.
  • a subsequent cycle of combination therapy is initiated the next day.
  • cycle 1 would occur during days 1-28
  • cycle 2 would occur during days 29-56
  • cycle 3 would occur during days 57-84, with no time period elapsing between the completion of a prior cycle and the initiation of a subsequent cycle of combination therapy.
  • a patient may be given a time period off between multiple cycles of combination therapy.
  • the length of this interruption between cycles of combination anti-CD40 antibody/bendamustine therapy will depend upon the health of the patient, history of disease progression, and responsiveness of the patient to the initial and/or prior cycles of combination anti-CD40 antibody/bendamustine therapy. Generally the length of this interruption is about 1 week to about 4 weeks, for example, about 1 week, about 2 weeks, about 3 weeks, or about 4 weeks.
  • combination therapy with these two anti-cancer agents comprises the administration of two therapeutically effective doses of bendamustine and at least one therapeutically effective dose of an anti-CD40 antibody to the patient in need thereof over each cycle of combination therapy.
  • bendamustine is administered on days 1 and 2 of the cycle, for example, on days 1 and 2 of a 21 -day, 28-day, or 35-day cycle of combination therapy.
  • a first therapeutically effective dose of anti-CD40i antibody is administered to the patient within 3 days of the start of a given cycle of combination therapy (for example, on day 1, 2, 3, or 4 of that cycle), and a second therapeutically effective dose of the antibody is optionally administered to the patient about 12-14 days following the administration of the first dose of anti-CD40 antibody (i.e., on day 13, 14, 15, or 16 of that cycle).
  • the agents may be administered simultaneously or sequentially, in either order.
  • the anti-CD40 antibody is administered prior to administering the bendamustine.
  • the combination therapy of the invention comprises administration of these two anti-cancer agents to a patient in need of treatment for a disease or condition associated with neoplastic B-cell growth, where the administration occurs over multiple cycles, each of which consists of about 28 days (4 weeks).
  • the combination therapy comprises administering a therapeutically effective dose of bendamustine to the patient on each of days 1 and 2 of the cycle, and
  • An exemplary dosing regimen for each of said 28-day cycles of combination therapy comprises the administration of a therapeutically effective dose of bendamustine on each of days 1 and 2 of said 28-day cycle, and administration of a first therapeutically effective dose of said anti-CD40 antibody (such as HCD122) within the first three days of the 28- day cycle, for example, on day 1, day 2, or day 3 of the 28-day cycle.
  • the first therapeutically effective dose of the anti-CD40 antibody (such as HCD122) is administered on day 1 of the 28-day cycle of combination therapy, and a second therapeutically effective dose of the anti-CD40 antibody is administered on day 15 of the 28-day cycle of combination therapy; in other embodiments, the first therapeutically effective dose of the anti-CD40 antibody (such as HCD122) is administered on day 2 of the 28-day cycle of combination therapy, and a second therapeutically effective dose of the anti-CD40 antibody is administered on day 15 of the 28-day cycle of combination therapy; in yet other embodiments, the first therapeutically effective dose of the anti-CD40 antibody (such as HCD122) is administered on day 3 of the 28-day cycle of combination therapy, and a second therapeutically effective dose of the anti-CD40 antibody is administered on day 15 of the 28-day cycle of combination therapy.
  • the first therapeutically effective dose of the anti-CD40 antibody (such as HCD122) is administered on day 1 of the 28-day cycle of combination therapy, and a second therapeutically effective dose of the anti
  • an alternative anti-CD40 antibody dosing schedule can be implemented, wherein the second therapeutically effective dose of the anti-CD40 antibody is withheld (i.e., the day 15 antibody dose is not administered).
  • a patient in need of treatment for a disease or condition associated with neoplastic B-cell growth is administered 3-8 cycles of combination anti-CD40 antibody/bendamustine therapy, each of which consists of a 28-day cycle, wherein a therapeutically effective dose of bendamustine in the range of about 60 mg/m 2 to about 120 mg/m 2 (for example, 60, 65, 70, 75, 80, 85, 90, 95, 100, 105, 1 10, 1 15, 120 mg/m 2 , or other such value falling between about 60 mg/m 2 and about 120 mg/m 2 ) is administered intravenously (iv) on day 1 and day 2 of each 28-day cycle, and a therapeutically effective dose of an anti-CD40 antibody (such as HCD122) in the range of about 2.5 mg/kg to about 5.0 mg/kg (body weight)(for example, about 2.5, 3.0, 3.5, 4.0 4.5
  • an anti-CD40 antibody such as HCD122
  • the patient is administered 6 cycles of this combination therapy according to this dosing regimen.
  • the therapeutically effective dose of bendamustine is about 90 mg/m 2 and the therapeutically effective dose of the anti-CD40 antibody (such as HCD122) is about 3.0 mg/kg, 3.5 mg/kg, 4.0 mg/kg, or 4.5 mg/kg, which are administered according to the foregoing dosing regimen for six (6) 28-day cycles.
  • these multiple cycles of combination therapy are followed by a maintenance cycle of anti-CD40 antibody therapy, where the anti-CD40 antibody (such as HCD122) is administered bi-weekly for up to three months at the same dose that was administered in the final cycle of combination anti-CD40 antibody/bendamustine therapy.
  • the patient undergoing multiple cycles of this combination anti-CD40 antibody/bendamustine therapy has a B-cell lymphoma that is refractory to rituximab and has received at least one prior chemotherapeutic regimen, as described elsewhere herein.
  • the antibody and bendamustine therapies are administered at the same time, they may be administered as a single pharmaceutical formulation or as two separate pharmaceutical formulations. When the therapies are not administered at the same time, they are administered as two separate pharmaceutical formulations.
  • this pharmaceutical formulation may be obtained by a method comprising the steps of (i) obtaining a lyophilized anti-CD40 antibody composition, (ii) obtaining a composition comprising bendamustine in a sterile diluent, and (iii) reconstituting the lyophilized antibody composition using the composition comprising bendamustine.
  • the invention therefore provides a pharmaceutical composition comprising (i) bendamustine, (ii) an anti-CD40 antibody, and (iii) a pharmaceutically acceptable carrier or excipient.
  • the invention also provides the use of (i) bendamustine and (ii) an anti-CD40 antibody, in the manufacture of a medicament for treating a human patient for a disease or condition associated with neoplastic B-cell growth.
  • the invention provides the use of (i) bendamustine and (ii) an anti-CD40 antibody, in the manufacture of two separate medicaments for treating a human patient for a disease or condition associated with neoplastic B-cell growth by combination therapy.
  • the invention also provides a kit for treating a human patient for a disease or condition associated with neoplastic B-cell growth, said kit comprising (i) bendamustine and (ii) an anti-CD40 antibody.
  • the kit may further comprise one or more devices for administering the combination therapy to a human patient, such as one or more of (i) a sterile needle and syringe, (ii) a sterile container ⁇ e.g., a glass bottle, plastic bottle or plastic bag) and drip chamber, (iii) a sterile tube with a regulating clamp, and (iv) a catheter.
  • the invention provides a method for treating a human patient for a disease or condition associated with neoplastic B-cell growth, said method comprising
  • the invention also provides a method for treating a human patient for a disease or condition associated with neoplastic B-cell growth, said method comprising administering to said patient an anti-CD40 antibody, wherein the patient has been pre-treated with bendamustine.
  • the invention further provides the use of an anti-CD40 antibody in the manufacture of a medicament for treating a human patient for a disease or condition associated with neoplastic B-cell growth, wherein said human patient has been pre- treated with bendamustine.
  • the invention also provides the use of bendamustine in the manufacture of a medicament for treating a human patient for a disease or condition associated with neoplastic B-cell growth, wherein said human patient has been pre- treated with an anti-CD40 antibody.
  • pre-treated or “pre-treatment” is intended the subject has received one or more doses of a first therapy prior to a second therapy.
  • Pre-treated or “pre-treatment” includes patients that have been treated with a first therapy within 2 years, within 18 months, within 1 year, within 6 months, within 2 months, within 6 weeks, within 1 month, within 4 weeks, within 3 weeks, within 2 weeks, within 1 week, within 6 days, within 5 days, within 4 days, within 3 days, within 2 days, or within 1 day prior to initiation of treatment with a second therapy.
  • pre-treated or “pre-treatment” thus includes patients that have been treated with an anti-CD40 antibody within 2 years, within 18 months, within 1 year, within 6 months, within 2 months, within 6 weeks, within 1 month, within 4 weeks, within 3 weeks, within 2 weeks, within 1 week, within 6 days, within 5 days, within 4 days, within 3 days, within 2 days, or within 1 day prior to initiation of treatment with the bendamustine therapy.
  • pre-treated or “pre-treatment” also includes patients that have been treated with the bendamustine therapy within 2 years, within 18 months, within 1 year, within 6 months, within 2 months, within 6 weeks, within 1 month, within 4 weeks, within 3 weeks, within 2 weeks, within 1 week, within 6 days, within 5 days, within 4 days, within 3 days, within 2 days, or within 1 day, prior to initiation of treatment with an anti-CD40 antibody.
  • Patients who have been pre-treated with an anti-CD40 antibody can be distinguished from other patients, e.g., by consulting patients' medical records or carrying out suitable in vitro test(s).
  • Patients who have been pre-treated with bendamustine can be distinguished from other patients, e.g., by consulting patients' medical records or carrying out suitable in vitro test(s).
  • the invention also provides the use of an anti-CD40 antibody in the manufacture of a medicament for treating a human patient for a disease or condition associated with neoplastic B-cell growth, wherein the medicament is administered prior to administration of bendamustine.
  • the invention provides the use of an anti- CD40 antibody in the manufacture of a medicament for treating a human patient for a disease or condition associated with neoplastic B-cell growth, wherein the medicament is administered subsequent to administration of bendamustine.
  • the invention also provides the use of bendamustine in the manufacture of a medicament for treating a human patient for a disease or condition associated with neoplastic B-cell growth, wherein the medicament is administered prior to administration of an anti-CD40 antibody.
  • the invention provides the use of bendamustine in the manufacture of a medicament for treating a human patient for a disease or condition associated with neoplastic B-cell growth, wherein the medicament is administered subsequent to administration of an anti-CD40 antibody.
  • the invention also provides an anti-CD40 antibody and bendamustine for simultaneous, separate, or sequential use in treating a human patient for a disease or condition associated with neoplastic B-cell growth by combination therapy.
  • the invention also provides the use of an anti-CD40 antibody in the manufacture of a medicament for simultaneous or sequential use in combination with bendamustine for treating a human patient for a disease or condition associated with neoplastic B-cell growth.
  • the invention also provides the use of bendamustine in the manufacture of a medicament for simultaneous or sequential use in combination with an anti-CD40 antibody for treating a human patient for a disease or condition associated with neoplastic B-cell growth.
  • the methods of the invention may comprise administering a dose of an anti-CD40 antibody at any time during a first or subsequent cycle of the bendamustine therapy.
  • the methods of the invention may comprise administering a dose of an anti- CD40 antibody between cycles of bendamustine therapy.
  • the combination therapy provides a synergistic improvement in therapeutic efficacy relative to the individual therapeutic agents when administered alone.
  • the term "synergy" is used to describe a combined effect of two or more active agents that is greater than the sum of the individual effects of each respective active agent.
  • the combined effect of two or more agents results in “synergistic inhibition" of an activity or process, for example, tumor growth
  • the inhibition of the activity or process is greater than the sum of the inhibitory effects of each respective active agent.
  • the term “synergistic therapeutic effect” therefore refers to a therapeutic effect observed with a combination of two or more therapies; wherein the therapeutic effect (as measured by any of a number of parameters, e.g., tumour growth delay) is greater than the sum of the individual therapeutic effects observed with the respective individual therapies.
  • the invention involves the use of anti-CD40 antibodies for the treatment of human patients having diseases or conditions associated with neoplastic B-cell growth.
  • TNF tumor necrosis factor
  • the first isoform (also known as the "long isoform” or “isoform 1 ”) is expressed as a 277- amino-acid precursor polypeptide (SEQ ID NO:9; first reported as GenBank Accession No. CAA43045, and identified as isoform 1 in GenBank Accession No. NP_001241), encoded by SEQ ID NO:8 (see GenBank Accession Nos. X60592 and NM_001250), which has a signal sequence represented by the first 19 residues.
  • the second isoform (also known as the "short isoform” or “isoform 2”) is expressed as a 203-amino-acid precursor polypeptide (SEQ ID NO:7; GenBank Accession No.
  • SEQ ID NO:6 GenBank Accession No. NM_152854
  • SEQ ID NO:6 GenBank Accession No. NM_152854
  • the precursor polypeptides of these two isoforms of human CD40 share in common their first 165 residues ⁇ i.e., residues 1-165 of SEQ ID NO:7 and SEQ ID NO:9).
  • the precursor polypeptide of the short isoform (shown in SEQ ID NO:7) is encoded by a transcript variant (SEQ ID NO:6) that lacks a coding segment, which leads to a translation frame shift; the resulting CD40 isoform contains a shorter and distinct C-terminus (residues 166-203 of SEQ ID NO:7) from that contained in the long isoform of CD40 (C-terminus shown in residues 166-277 of SEQ ID NO:9).
  • the term "CD40,” or "CD40 antigen,” “CD40 cell surface antigen,” or "CD40 receptor” encompasses both the short and long isoforms of CD40.
  • CD40-expressing cells any normal or malignant cells that express detectable levels of the CD40 antigen.
  • Methods for detecting CD40 antigen expression in cells are well known in the art and include, but are not limited to, PCR techniques, immunohistochemistry, flow cytometry, Western blot, ELISA, and the like. These methods allow for the detection of CD40 mR A, CD40 antigen and cell-surface CD40 antigen.
  • the CD40-expresing cells are cells that express detectable levels of cell-surface CD40 antigen.
  • CD40 ligand or “CD40L” is intended the 32-33 kDa transmembrane protein that also exists in two smaller biologically active soluble forms, 18 kDa and 31 kDa, respectively (Graf el al. (1995) Eur. J. Immunol. 25: 1749-1754; Mazzei et al. (1995) J. Biol Chem. 270:7025-7028; Pietravalle et al. (1996) J Biol. Chem. 271 :5965-5967). Human CD40L is also known as CD154 or gp39.
  • human patient is intended a human who is afflicted with, at risk of developing or relapsing with, any disease or condition associated with neoplastic B-cell growth.
  • disease or condition associated with neoplastic B-cell growth is intended any disease or condition (including pre-malignant conditions) involving uncontrolled growth of cells of B-cell lineage.
  • diseases and conditions include, but are not limited to, acute lymphoblastic leukemia (ALL), acute myelogenous leukemia (AML), chronic myelogenous leukemia (CML), chronic lymphocytic leukemia (CLL), prolymphocytic leukemia (PLL), small lymphocytic leukemia (SLL), diffuse small lymphocytic leukemia (DSLL), diffuse large B-cell lymphoma (DLBCL), hairy cell leukemia, non-Hodgkin's lymphomas, including follicular lymphoma, Hodgkin's disease, Epstein-Barr Virus (EBV) induced lymphomas, myelomas such as multiple myeloma, Waldenstrom's macroglobulinemia, heavy chain disease, mucosal associated lymphoi
  • the methods of the invention find use in the treatment of subjects having non- Hodgkin's lymphomas related to abnormal B cell proliferation or accumulation.
  • lymphomas will be referred to according to the Working Formulation classification scheme, that is those B cell lymphomas categorized as low grade, intermediate grade, and high grade (see "The Non-Hodgkin's Lymphoma Pathologic Classification Project," Cancer 49(1982):21 12-2135).
  • low-grade B cell lymphomas include small lymphocytic, follicular small-cleaved cell, and follicular mixed small-cleaved and large cell lymphomas; intermediate-grade lymphomas include follicular large cell, diffuse small cleaved cell, diffuse mixed small and large cell, and diffuse large cell lymphomas; and high-grade lymphomas include large cell
  • the methods of the invention can be used to treat low-, intermediate-, and high-grade B cell lymphomas.
  • the methods of the invention are useful in the therapeutic treatment of B cell lymphomas that are classified according to the Revised European and American
  • B cell lymphomas include, but are not limited to, lymphomas classified as precursor B cell neoplasms, such as B lymphoblastic leukemia/lymphoma; peripheral B cell neoplasms, including B cell chronic lymphocytic leukemia/small lymphocytic lymphoma, lymphoplasmacytoid
  • lymphoma/immunocytoma mantle cell lymphoma (MCL), follicle center lymphoma (follicular lymphoma) (including diffuse small cell, diffuse mixed small and large cell, and diffuse large cell lymphomas), marginal zone B-cell lymphoma (including extranodal, nodal, and splenic types, e.g., extranodal marginal zone B-cell lymphoma of mucosa-associated lymphoid tissue), plasmacytoma/ myeloma, diffuse large cell B cell lymphoma of the subtype primary mediastinal (thymic), Burkitt's lymphoma, and Burkitt's like high-grade B cell lymphoma; and unclassifiable low-grade or high-grade B cell lymphomas.
  • MCL mantle cell lymphoma
  • follicle center lymphoma follicular lymphoma
  • marginal zone B-cell lymphoma including extranodal,
  • combination therapy is used to provide a positive therapeutic response with respect to a disease or condition.
  • positive therapeutic response is intended an improvement in the disease or condition, and/or an improvement in the symptoms associated with the disease or condition, as a result of the therapeutic activity of the combination therapy. That is, an anti-proliferative effect, the prevention of further tumor outgrowths, a reduction in tumor size, a reduction in the number of neoplastic cells, and/or a decrease in one or more symptoms associated with CD40- expressing cells can be observed.
  • a positive therapeutic response would refer to one or more of the following improvements in the disease: (1) a reduction in tumor size; (2) a reduction in the number of neoplastic cells; (3) an increase in neoplastic cell death; (4) inhibition of neoplastic cell survival; (4) inhibition (i.e., slowing to some extent, preferably halting) of tumor growth; (5) inhibition (i.e., slowing to some extent, preferably halting) of neoplastic cell infiltration into peripheral organs; (6) inhibition (i.e., slowing to some extent, preferably halting) of tumor metastasis; (7) the prevention of further tumor outgrowths; (8) an increased patient survival rate; and (9) some relief from one or more symptoms associated with the disease or condition.
  • Positive therapeutic responses in any given disease or condition can be determined by standardized response criteria specific to that disease or condition.
  • Tumor response can be assessed for changes in tumor morphology (i.e., overall tumor burden, tumor size, and the like) using screening techniques such as magnetic resonance imaging (MRI) scan, x-radiographic imaging, computed tomographic (CT) scan, bone scan imaging, endoscopy, and tumor biopsy sampling including bone marrow aspiration (BMA) and counting of tumor cells in the circulation.
  • MRI magnetic resonance imaging
  • CT computed tomographic
  • BMA bone marrow aspiration
  • the subject undergoing therapy may experience the beneficial effect of an improvement in the symptoms associated with the disease.
  • the subject may experience a decrease in the so-called B symptoms, i.e., night sweats, fever, weight loss, and/or urticaria.
  • therapy with an anti-CD40 therapeutic agent may block and/or prolong the time before development of a related malignant condition, for example, development of multiple myeloma in subjects suffering from monoclonal gammopathy of undertermined significance (MGUS).
  • MGUS monoclonal gammopathy of undertermined significance
  • An improvement in the disease may be characterized as a complete response.
  • complete response is intended an absence of clinically detectable disease with normalisation of any previously abnormal radiographic studies, bone marrow, and cerebrospinal fluid (CSF) or abnormal monoclonal protein in the case of myeloma. Such a response may persist for at least 4 to 8 weeks, or sometimes 6 to 8 weeks, following treatment according to the methods of the invention. Alternatively, an improvement in the disease may be categorized as being a partial response.
  • partial response is intended at least about a 50% decrease in all measurable tumor burden (i.e., the number of malignant cells present in the subject, or the measured bulk of tumor masses or the quantity of abnormal monoclonal protein) in the absence of new lesions, which may persist for 4 to 8 weeks, or 6 to 8 weeks.
  • an effective amount or “therapeutically or prophylactically effective amount” is intended an amount of antibody therapy or bendamustine therapy that, when administered as a part of a combination therapy, brings about a positive therapeutic response with respect to patient treatment. Suitable amounts are described in more detail elsewhere herein.
  • Tumor refers to all neoplastic cell growth and proliferation, whether malignant or benign, and all pre-cancerous and cancerous cells and tissues.
  • Neoplastic refers to any form of dysregulated or unregulated cell growth, whether malignant or benign, resulting in abnormal growth.
  • Neoplastic cells include malignant and benign cells having dysregulated or unregulated cell growth.
  • cancer and “cancerous” refer to or describe the physiological condition in mammals that is typically characterized by unregulated cell growth.
  • Treatment is herein defined as the application or administration of combination therapy to a patient, or application or administration of combination therapy to an isolated tissue from a patient, where the patient has a disease, a symptom of a disease, or a predisposition toward a disease, where the purpose is to cure, heal, alleviate, relieve, alter, remedy, ameliorate, improve, or affect the disease, the symptoms of the disease, or the predisposition toward the disease.
  • the methods of the invention are particularly useful for treating patients who have previously been administered other oncotherapeutic treatments.
  • the methods of the invention are useful for treating a disease or condition associated with neoplastic B-cell growth, including the B cell lymphomas listed above, that are refractory to (i.e., resistant to, or have become resistant to) first-line oncotherapeutic treatments.
  • oncotherapeutic or “oncotherapy” is intended to mean a treatment for cancer such as chemotherapy, surgery, radiation therapy, single anti-cancer antibody therapy, and combinations thereof.
  • Such oncotherapy can include chemotherapy regimens such as treatment with CVP (cyclophosphamide, vincristine and prednisone), R-CVP (rituximab plus CVP), CHOP (cyclophosphamide, doxorubicin, vincristine and prednisone), R-CHOP
  • CVP cyclophosphamide, vincristine and prednisone
  • R-CVP rituximab plus CVP
  • CHOP cyclophosphamide, doxorubicin, vincristine and prednisone
  • R-CHOP cyclophosphamide, doxorubicin, vincristine and prednisone
  • such oncotherapies can include radiation treatment, including
  • myleoablative therapies or radioimmunotherapy, for example, with yttrium-90-labeled ibritumomab tiuxetan or iodine- 131 -labeled tositumomab.
  • the combination therapy of the invention may be useful for treating a human patient who has previously been administered (i) anti-CD40 antibody therapy alone, (ii) bendamustine therapy alone, (iii) a chemotherapeutic regimen alone, (iv) anti- CD20 antibody therapy (such as the chimeric anti-CD20 antibody rituximab) alone, or (iv) an anti-CD20 antibody-containing regimen (such as a ntuximab-containing regimen), particularly combination therapy with CHOP and an anti-CD20 antibody (such as rituximab, wherein the combination therapy is commonly termed R-CHOP) or combination therapy with CVP and an anti-CD20 antibody (jsuch as rituximab, wherein the combination therapy is commonly termed R-CVP).
  • anti-CD20 antibody therapy such as the chimeric anti-CD20 antibody rituximab
  • an anti-CD20 antibody-containing regimen such as a ntuximab-containing regimen
  • R-CHOP anti
  • the invention may be particularly useful for treating diseases or conditions that are refractory to therapy with other oncotherapeutic treatments.
  • the invention may therefore be useful in treating diseases or conditions that are refractory to therapy with (i) a chemotherapeutic regimen alone (such as CHOP), (ii) an anti-CD40 antibody (such as HCD122) alone, (iii) an anti-CD20 antibody (such as rituximab) alone, or (iv) combination therapy with CHOP and an anti-CD20 antibody (for example, R-CHOP) or CVP and an anti-CD20 antibody (for example, R-CVP).
  • a chemotherapeutic regimen such as CHOP
  • an anti-CD40 antibody such as HCD122
  • an anti-CD20 antibody such as rituximab
  • combination therapy with CHOP and an anti-CD20 antibody for example, R-CHOP
  • CVP anti-CD20 antibody
  • R-CVP anti-CD20 antibody
  • a disease or condition can be refractory to therapy with a particular therapeutic agent either from the onset of treatment with the particular therapeutic agent ⁇ i.e., non-responsive to initial exposure to the therapeutic agent), or as a result of developing resistance to the therapeutic agent, either over the course of a first treatment period with the therapeutic agent or during a subsequent treatment period with the therapeutic agent.
  • the invention therefore provides methods, compositions, uses and kits for treating a human patient for a disease or condition associated with neoplastic B- cell growth, wherein said disease or condition is refractory to an oncotherapeutic treatment other than the combination therapy of the invention.
  • the invention may also be particularly useful for treating patients who have relapsed after therapy with other oncotherapeutic treatments.
  • the invention may therefore be useful in treating patients who have relapsed after therapy with (i) CHOP alone, (ii) an anti-CD40 antibody (such as HCD122) alone, (iii) bendamustine alone, (iv) an anti-CD20 antibody (such as rituximab) alone, or (v) combination therapy with CHOP and an anti- CD20 antibody (R-CHOP).
  • relapsed is meant that the patient achieved a partial or complete response to a prior oncotherapeutic treatment, but has subsequently had a recurrence of the disease or condition.
  • the invention therefore provides methods, compositions, uses and kits for treating a human patient for a disease or condition associated with neoplastic B-cell growth, wherein said patient has relapsed after therapy with an oncotherapeutic treatment other than the combination therapy of the invention.
  • the combination therapy of the invention addresses problems associated with therapy using rituximab (the IDEC-C2B8 monoclonal antibody (Biogen pout or
  • Rituximab is a chimeric anti-CD20 monoclonal antibody containing human IgGl and kappa constant regions with murine variable regions isolated from a murine anti-CD20 monoclonal antibody (Reff et al (1994) Blood 83:435-445).
  • the methods of the invention enable the treatment of patients having a disease or condition associated with CD40-expressing B- cells, which might otherwise have been treated with rituximab or by combination therapy with rituximab and chemotherapeutic agents (e.g., CHOP, CVP).
  • chemotherapeutic agents e.g., CHOP, CVP
  • the invention also provides methods, compositions, uses, and kits for treating a human patient for a disease or condition associated with neoplastic B-cell growth, including the B cell lymphomas listed above, by combination therapy, wherein the patient has previously been administered the chimeric anti-CD20 antibody rituximab, or has previously been administered rituximab and has received at least one prior chemotherapeutic regimen.
  • the invention may be useful in treating patients with a disease or condition associated with neoplastic B-cell growth that is refractory to therapy with (i) rituximab alone, or (ii) combination therapy with rituximab and a
  • chemotherapeutic regimen for example, CHOP and rituximab (R-CHOP) or CVP and rituximab (R-CVP)
  • the invention may also be useful in treating patients who have relapsed after therapy with (i) rituximab alone, or (ii) combination therapy with rituximab and a chemotherapeutic regimen (for example, CHOP and rituximab (R-CHOP) or CVP and rituximab (R-CVP)).
  • the methods, compositions, uses, and kits are directed to treatment of patients for a non-Hodgkin's lymphoma, including CD40 + follicular lymphoma, where this disease has progressed during or within 6 months of treatment with rituximab or a rituximab-containing regimen and who have received treatment with at least one prior chemotherapeutic regimen, for example, CHOP, CVP, or a fiudarabine-based chemotherapy.
  • chemotherapeutic regimen for example, CHOP, CVP, or a fiudarabine-based chemotherapy.
  • Patients who have been pre-treated with rituximab can be distinguished from other patients, e.g., by consulting patients' medical records Or carrying out suitable in vitro test(s). For example, the number of circulating CD 19 + B-cells is depleted in patients treated with rituximab, and numbers of circulating CD19 + B-cells can be monitored using suitable methods, e.g., FACS (McLaughlin et al. (1998) J. Clin. Oncol. 16(8):2825-2833; Maloney et al. (1997) Blood 90(6):2 ⁇ 88-2195).
  • suitable methods e.g., FACS (McLaughlin et al. (1998) J. Clin. Oncol. 16(8):2825-2833; Maloney et al. (1997) Blood 90(6):2 ⁇ 88-2195).
  • Natural antibodies are usually heterotetrameric glycoproteins of about 150,000 daltons, composed of two identical light (L) chains and two identical heavy (H) chains. Each light chain is linked to a heavy chain by one covalent disulfide bond, while the number of disulfide linkages varies among the heavy chains of different isotypes. Each heavy and light chain also has regularly spaced intrachain disulfide bridges. Each heavy chain has at one end a variable domain (VH) followed by a number of constant domains.
  • VH variable domain
  • Each light chain has a variable domain at one end (V L ) and a constant domain at its other end; the constant domain of the light chain is aligned with the first constant domain of the heavy chain, and the light chain variable domain is aligned with the variable domain of the heavy chain. Particular amino acid residues are believed to form an interface between the light and heavy-chain variable domains.
  • the term "variable” refers to the fact that certain portions of the variable domains differ extensively in sequence among antibodies. The variable regions confer antigen-binding specificity.
  • the constant domains are not involved directly in binding an antibody to an antigen, but exhibit various effector functions, such as Fc receptor (FcR) binding, participation of the antibody in antibody-dependent cellular toxicity, initiation of complement dependent cytotoxicity, and mast cell degranulation.
  • Fc receptor Fc receptor
  • the "light chains" of antibodies from any vertebrate species can be assigned to one of two clearly distinct types, called kappa ( ⁇ ) and lambda ( ⁇ ), based on the amino acid sequences of their constant domains.
  • antibodies can be assigned to different classes. There are five major classes of human antibodies: IgA, IgD, IgE, IgG, and IgM, and several of these may be further divided into subclasses (isotypes), e.g., IgGl, IgG2, IgG3, IgG4, IgAl, and IgA2.
  • the heavy-chain constant domains that correspond to the different classes of antibodies are called alpha, delta, epsilon, gamma, and mu, respectively.
  • the subunit structures and three-dimensional configurations of different classes of antibodies are well known. Different isotypes have different effector functions. For example, human IgGl and IgG3 isotypes have ADCC (antibody dependent cell-mediated cytotoxicity) activity.
  • IgGl antibodies, in particular human IgG 1 antibodies are particularly useful in the methods of the invention.
  • Human effector cells are leukocytes that express one or more FcRs and perform effector functions. Preferably, the cells express at least FcyRIII and carry out antigen- dependent cell-mediated cyotoxicity (ADCC) effector function.
  • ADCC antigen- dependent cell-mediated cyotoxicity
  • human leukocytes that mediate ADCC include peripheral blood mononuclear cells (PBMC), natural killer (NK) cells, monocytes, macrophages, eosinophils, and neutrophils, with PBMCs and NK cells being preferred.
  • Antibodies that have ADCC activity are typically of the IgGl or IgG3 isotype. Note that in addition to isolating IgGl and IgG3 antibodies, ADCC-mediating antibodies can be made by combining a variable region from a non- ADCC antibody with an IgGl or IgG3 isotype constant region.
  • Fc receptor or “FcR” are used to describe a receptor that binds to the Fc region of an antibody.
  • the preferred FcR is a native-sequence human FcR.
  • a preferred FcR is one that binds an IgG antibody (a gamma receptor) and includes receptors of the FcyRI, FcyRII, and FcyRIII subclasses, including allelic variants and alternatively spliced forms of these receptors.
  • FcyRII receptors include FcyRIIA (an “activating receptor") and FcyRIIB (an “inhibiting receptor”), which have similar amino acid sequences that differ primarily in the cytoplasmic domains thereof.
  • Activating receptor FcyRIIA contains an immunoreceptor tyrosine-based activation motif (ITAM) in its cytoplasmic domain.
  • Inhibiting receptor FcyRIIB contains an immunoreceptor tyrosine-based inhibition motif (ITIM) in its cytoplasmic domain (see Daeron (1997) Annu. Rev. Immunol. 15:203-234).
  • FcRs are reviewed in Ravetch and Kinet (1991) Annu. Rev. Immunol. 9:457-492 (1991); Capel et al. (1994) Immunomethods 4:25-34; and de Haas et al. (1995) J. Lab. Clin. Med. 126:330-341.
  • FcR FcR
  • FcRn neonatal receptor
  • antibody is used herein in the broadest sense and covers fully assembled antibodies, antibody fragments which retain the ability to specifically bind to the CD40 antigen (e.g., Fab, F(ab')2> Fv, and other fragments), single chain antibodies (scFv), diabodies, bispecific antibodies, chimeric antibodies, humanized antibodies, fully human antibodies, and the like, and recombinant peptides comprising the foregoing.
  • CD40 antigen e.g., Fab, F(ab')2> Fv, and other fragments
  • scFv single chain antibodies
  • diabodies bispecific antibodies
  • chimeric antibodies humanized antibodies
  • fully human antibodies fully human antibodies, and the like, and recombinant peptides comprising the foregoing.
  • antibody covers both polyclonal and monoclonal antibodies.
  • anti-CD40 antibody encompasses any antibody that specifically recognizes the CD40 antigen.
  • anti-CD40 antibodies for use in the methods of the present invention exhibit a strong single-site binding affinity for the CD40 antigen.
  • Such monoclonal antibodies exhibit an affinity for CD40 (K D ) of at least 10 "5 M, preferably at least 10 "6 M, at least 10 " 7 M, at least 10 "8 M, at least 10 "9 M, at least 10 "10 M, at least 10 " " M or at least 10 "12 M, when measured using a standard assay such as BiacoreTM.
  • Biacore analysis is known in the art and details are provided in the "BIAapplications handbook”.
  • the anti- CD40 antibody binds to the CD40 antigen on the surface of human B-cells, but does not bind to a significant extent other antigens on the surface of human B-cells, such as the CD20 antigen.
  • anti-CD40 antibodies for use in the methods of the present invention can be produced using any suitable antibody production method known to those of skill in the art.
  • the anti-CD40 antibody used in the methods of the present invention may be a monoclonal antibody.
  • the term "monoclonal antibody” (and “mAb”) as used herein refers to an antibody obtained from a substantially homogeneous population of antibodies, i.e., the individual antibodies comprising the population are identical except for possible naturally occurring mutations that may be present in minor amounts. The term is not limited regarding the species of the antibody and does not require production of the antibody by any particular method.
  • polyclonal antibody preparations which typically include different antibodies directed against different antigenic determinants (epitopes)
  • each monoclonal antibody is directed against a single determinant (epitope) on the antigen.
  • monoclonal as originally used in relation to antibodies referred to antibodies produced by a single clonal line of immune cells, as opposed to “polyclonal” antibodies that, while all recognizing the same target protein, were produced by different B cells and would be directed to different epitopes on that protein.
  • the word “monoclonal” does not imply any particular cellular origin, but refers to any population of antibodies that all have the same amino acid sequence and recognize the same epitope in the same target protein.
  • a monoclonal antibody may be produced using any suitable protein synthesis system, including immune cells, non-immune cells, acellular systems, etc.
  • the product datasheets for the CDR-grafted humanized antibody SynagisTM expressed in a murine myeloma NSO cell line, the humanized antibody HerceptinTM expressed in a CHO cell line, and the phage- displayed antibody HumiraTM expressed in a CHO cell line all refer to the products as monoclonal antibodies.
  • Epitope is intended the part of an antigenic molecule to which an antibody is produced and to which the antibody will bind.
  • Epitopes can comprise linear amino acid residues (i.e., residues within the epitope are arranged sequentially in a linear fashion), non-linear amino acid residues (referred to herein as “non-linear epitopes”; these epitopes are not arranged sequentially), or both linear and non-linear amino acid residues.
  • Monoclonal antibodies may be made by the hybridoma method first described by Kohler et al (1975) Nature 256:495, or may be made by recombinant DNA methods (see, e.g., U.S. Patent No. 4,816,567). Monoclonal antibodies may also be isolated from antibody phage libraries generated using the techniques described in, for example, McCafferty et al. (1990) Nature 348:552-554 (1990) and U.S. Patent No. 5,514,548. Clackson et al. (1991) Nature 352:624-628 and Marks et al. (1991) 7. Mol. Biol.
  • the DNA encoding the monoclonal antibodies is readily isolated and sequenced using conventional procedures. Once isolated, the DNA may be placed into expression vectors, which are then transfected into host cells such as E. coli cells, simian COS cells, Chinese Hamster Ovary (CHO) cells, or myeloma cells that do not otherwise produce immunoglobulin protein, to obtain the synthesis of monoclonal antibodies in the recombinant host cells.
  • host cells such as E. coli cells, simian COS cells, Chinese Hamster Ovary (CHO) cells, or myeloma cells that do not otherwise produce immunoglobulin protein
  • antibody can be produced in a cell line such as a CHO cell line, as disclosed in U.S. Patent Nos. 5,545,403; 5,545,405; and 5,998,144. Briefly the cell line is transfected with vectors capable of expressing a light chain and a heavy chain, respectively. By transfecting the two proteins on separate vectors, chimeric antibodies can be produced. Another advantage is the mammalian glycosylation of the antibody in CHO cells. CHO cells are a preferred source of recombinant antibodies for use in the combination therapy of the invention.
  • a "host cell,” as used herein, refers to a microorganism or a eukaryotic cell or cell line cultured as a unicellular entity that can be, or has been, used as a recipient for a recombinant vector or other transfer polynucleotides, and include the progeny of the original cell that has been transfected. It is understood that the progeny of a single cell may not necessarily be completely identical in morphology or in genomic or total DNA complement as the original parent, due to natural, accidental, or deliberate mutation.
  • Monoclonal antibodies to CD40 are known in the art. See, for example, the sections dedicated to B-cell antigen in McMichael, ed. (1987; 1989) Leukocyte Typing III and IV (Oxford University Press, New York); U.S. Patent Nos. 5,674,492; 5,874,082; 5,677,165; 6,056,959; WO 00/63395; International Publication Nos. WO 02/28905 and WO 02/28904; Gordon et al. (1988) J. Immunol. 140:1425; Valle et al. (1989) Eur. J. Immunol 19:1463; Clark et al. (1986) PNAS 83:4494; Paulie et al.
  • chimeric antibodies encompasses chimeric antibodies.
  • chimeric antibodies antibodies that are most preferably derived using recombinant DNA techniques and which comprise both human (including immunologically "related" species, e.g., chimpanzee) and non-human components.
  • the constant region of the chimeric antibody is most preferably substantially identical to the constant region of a natural human antibody; the variable region of the chimeric antibody is most preferably derived from a non-human source and has the desired antigenic specificity to CD40.
  • the non-human source can be any vertebrate source that can be used to generate antibodies to CD40 antigen.
  • non-human sources include, but are not limited to, rodents (e.g., rabbit, rat, mouse, etc.; see, for example, U.S. Patent No. 4,816,567) and non-human primates (e.g., Old World Monkey, Ape, etc.; see, for example, U.S. Patent Nos. 5,750,105 and 5,756,096).
  • the phrase "constant region” refers to the portion of the antibody molecule that confers effector functions.
  • mouse constant regions were substituted by human constant regions.
  • the constant regions of the subject humanized antibodies were derived from human antibodies. However, these antibodies can elicit an unwanted and potentially dangerous immune response in humans and there was a loss of affinity .
  • humanized antibodies encompasses humanized antibodies.
  • humanized antibodies are human antibodies (recipient antibody) in which residues from a hypervariable region (also known as complementarity determining region or CDR) of the recipient are replaced by residues from a hypervariable region of a non-human species (donor antibody) such as mouse, rat, rabbit, or nonhuman primate having the desired specificity, affinity, and capacity.
  • donor antibody such as mouse, rat, rabbit, or nonhuman primate having the desired specificity, affinity, and capacity.
  • complementarity determining region refers to amino acid sequences which together define the binding affinity and specificity of the natural Fv region of a native antibody binding site.
  • humanized antibodies may comprise residues that are not found in the recipient antibody or in the donor antibody. These modifications are made to further refine antibody performance (e.g. , to obtain desired affinity).
  • the humanized antibody will comprise substantially all of at least one, and typically two, variable domains, in which all or substantially all of the hypervariable regions correspond to those of a non-human antibody and all or substantially all of the framework regions are those of a human antibody sequence.
  • the humanized antibody optionally also will comprise at least a portion of an antibody constant region (Fc), typically that of a human antibody.
  • Fc antibody constant region
  • humanized antibodies may include antibodies wherein substantially less than an intact human variable domain has been substituted by the corresponding sequence from a non-human species.
  • humanized antibodies are typically human antibodies in which some CDR residues and possibly some framework residues are substituted by residues from analogous sites in rodent antibodies. See, for example, U.S. Patent Nos. 5,225,539; 5,585,089; 5,693,761 ; 5,693,762; 5,859,205. See also U.S. Patent No.
  • Humanized anti-CD40 antibodies can also be produced using the Humanized anti-CD40 antibodies.
  • Humanized anti-CD40 monoclonal antibodies include antibodies such as SGN-40
  • the present invention can also be practiced using xenogeneic or modified antibodies produced in a non-human mammalian host, more particularly a transgenic mouse, characterized by inactivated endogenous immunoglobulin (Ig) loci.
  • a transgenic mouse characterized by inactivated endogenous immunoglobulin (Ig) loci.
  • Ig immunoglobulin loci
  • competent endogenous genes for the expression of light and heavy subunits of host immunoglobulins are rendered non-functional and substituted with the analogous human immunoglobulin loci.
  • These transgenic animals produce human antibodies in the substantial absence of light or heavy host immunoglobulin subunits. See, for example, U.S. Patent Nos. 5,877,397 and 5,939,598.
  • Fully human antibodies to CD40 are obtained by immunizing transgenic mice.
  • One such mouse is obtained using
  • mice transgenic for the human IgGi heavy chain locus and the human ⁇ light chain locus were immunized with Sf9 cells expressing human CD40. Mice can also be transgenic for other isotypes.
  • the anti-CD40 antibody will have a light chain variable domain (VL) that comprises the light chain CDR sequences of HCD122.
  • VL light chain variable domain
  • the anti-CD40 antibody will have a light chain variable domain that comprises an amino acid sequence as shown in SEQ ID NO: 10 for CDR-L1, an amino acid sequence as shown in SEQ ID NO: l 1 for CDR-L2, and an amino acid sequence as shown in SEQ ID NO: 12 for CDR-L3.
  • the anti-CD40 antibody will have a heavy chain variable domain (VH) that comprises the heavy chain CDR sequences of HCD122.
  • the anti-CD40 antibody will have a heavy chain variable domain (VH) that comprises an amino acid sequence as shown in SEQ ID NO: 13 for CDR-H1, an amino acid sequence as shown in SEQ ID NO: 14 for CDR-H2, and an amino acid sequence as shown in SEQ ID NO: 15 for CDR-H3.
  • VH heavy chain variable domain
  • the anti-CD40 antibody will have a light chain variable domain (VL) that comprises the light chain CDR sequences of HCD122, and a heavy chain variable domain (V H ) that comprises the heavy chain CDR sequences of HCD122.
  • VL light chain variable domain
  • V H heavy chain variable domain
  • the anti-CD40 antibody will have a light chain variable domain (VL) that comprises an amino acid sequence as shown in SEQ ID NO: 10 for CDR-Ll, an amino acid sequence as shown in SEQ ID NO::l 1 for CDR-L2, and an amino acid sequence as shown in SEQ ID NO: 12 for CDR-L3, and a heavy chain variable domain ( ⁇ 3 ⁇ 4) that comprises an amino acid sequence as shown in SEQ ID NO: 13 for CDR-H1, an amino acid sequence as shown in SEQ ID NO: 14 for CDR-H2, and an amino acid sequence as shown in SEQ ID NO: 15 for CDR-H3.
  • the CDRs in a light chain variable region are amino acids 24-34 (CDR-Ll), 50-56 (CDR-L2) and 89-97 (CDR-L3)
  • the CDRs in a heavy chain variable region are amino acids 31-35 (CDR-H1), 50-65 (CDR-H2) and 95-102 (CDR- H3).
  • Another well-known scheme is the Chothia numbering scheme (Chothia & Lesk (1987) Mol. Biol. 196:901- 917).
  • the CDRs in a light chain variable region are amino acids 26-32 (CDR-Ll), 50-52 (CDR-L2) and 91-96 (CDR-L3)
  • the CDRs in a heavy chain variable region are amino acids 26-32 (CDR-H1), 53-55 (CDR-H2) and 96-101 (CDR-H3).
  • Antibody fragments comprise a portion of an intact antibody, preferably the antigen-binding or variable region of the intact antibody.
  • antibody fragments include Fab, F(ab')2, and Fv fragments.
  • Fab is intended a monovalent antigen-binding fragment of an antibody that contains the constant domain of the light chain and the first constant domain (CHI) of the heavy chain.
  • Papain digestion of antibodies produces two identical Fab fragments, and a residual "Fc” fragment, whose name reflects its ability to crystallize readily.
  • F(ab')2 is intended a bivalent antigen-binding fragment of an antibody that contains both light chains and part of both heavy chains, and which is retains the ability to cross-link antigen. Pepsin treatment yields an F(ab') 2 fragment.
  • Fv is the minimum antibody fragment that contains a complete antigen recognition and binding site.
  • This region consists of a dimer of one heavy- and one light-chain variable domain in tight, non- covalent association. It is in this configuration that the three CDRs of each variable domain interact to define an antigen-binding site on the surface of the VH-V l dimer. Collectively, the six CDRs confer antigen-binding specificity to the antibody. However, even a single variable domain (or half of an Fv comprising only three CDRs specific for an antigen) has the ability to recognize and bind antigen, although at a lower affinity than the entire binding site.
  • the invention may also use a single-chain Fv (scFv) ⁇ which is a polypeptide comprising the VH and V L domains of an antibody, wherein these domains are present in a single polypeptide chain (see e.g., U.S. Patents 4,946,778,: 5,260,203, 5,455,030, and 5,856,456).
  • scFv polypeptide comprises a polypeptide linker between the V H and V L domains that enables the scFv to form the desired structure for antigen binding.
  • Fragments of an anti-CD40 antibody are suitable for use in the methods of the invention so long as they retain the ability to bind to the CD40 antigen on the surface of human B-cells. Such fragments are referred to herein as "antigen-binding" fragments. Such fragments are preferably characterized by functional properties similar to the corresponding full-length antibody. Thus, for example, a fragment of a full-length anti- CD40 antibody will preferably be capable of specifically binding a human CD40 antigen expressed on the surface of a human cell, and is free of significant agonist activity as described elsewhere herein. Fragments of an anti-CD40 antibody for use in the methods of the invention may in some instances retain the ability to bind to the relevant FcR or FcRs.
  • F(ab')2 fragments can be isolated directly from recombinant host cell culture.
  • Other techniques for the production of antibody fragments will be apparent to the skilled practitioner.
  • the anti-CD40 antibodies used in the combination therapy of the invention are free of significant agonist activity when bound to CD40 antigen on the surface of human B-cells. In some embodiments, their binding to CD40 on the surface of human B-cells may result in inhibition of the proliferation and differentiation of the B-cells.
  • the anti- CD40 antibodies suitable for use in the methods of the invention include those antibodies that can exhibit antagonist activity toward human B-cells expressing the cell-surface CD40 antigen. These anti-CD40 antibodies and antigen-binding fragments thereof are referred to herein as "antagonist anti-CD40 antibodies.”
  • An "agonist” combines with a receptor on a cell and initiates a reaction or activity that is similar to or the same as that initiated by a natural ligand of the receptor.
  • An agonist of CD40 induces any or all of, but not limited to, the following responses: B cell proliferation and/or differentiation; upregulation of intercellular adhesion via such molecules as ICAM- 1 , E-selectin, VC AM, and the like; secretion of pro-inflammatory cytokines such as IL-1, IL-6, IL-8, IL-12, TNF, and the like; signal transduction through the CD40 receptor by such pathways as TRAF ⁇ e.g., TRAF2 and/or TRAF3), MAP kinases such as NIK (NF- ⁇ inducing kinase), I-kappa B kinases (IKK / ⁇ ), transcription factor NF- ⁇ , Ras and the MEK/ERK pathway, the PI3K AKT pathway, the P38 MAPK pathway, and the like; trans
  • significant agonist activity is intended an agonist activity of at least 30%, 10 35%, 40%, 45%, 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% greater than the agonist activity induced by a negative control as measured in an assay of a B cell response.
  • "significant" agonist activity is an agonist activity that is at least 2- fold greater or at least 3-fold greater than the agonist activity induced by a negative control as measured in an assay of a B cell response.
  • the B cell response of interest is B cell proliferation
  • "significant" agonist activity would be induction of a level of B cell proliferation that is at least 2-fold greater or at least 3-fold greater than the level of B cell proliferation induced by a negative control.
  • an antibody that does not bind to CD40 serves as the negative control.
  • a substance "free of significant agonist activity” would exhibit an agonist activity of not more than about 25% greater than the agonist activity induced by a negative control, preferably not more than about 20% greater, 15% greater, 10% greater, 5% greater, 1 % greater, 0.5% greater, or even not more than about 0.1% greater than the agonist activity induced by a negative control as measured in an assay of a B cell response.
  • An "antagonist" of CD40 prevents or reduces induction of any of the responses induced by binding of the CD40 receptor to an agonist ligand, particularly CD40L.
  • the antagonist may reduce induction of a response to CD40L binding by 5%, 10%, 15%, 20%, 25%, 30%, 35%, preferably 40%, 45%, 50%, 55%, 60%, more preferably 70%, 80%, 85%, and most preferably 90%, 95%, 99%, or 100%.
  • Preferred antibodies and fragments for use in the methods of the invention are anti-CD40 antibodies that are free of significant agonist activity when bound to CD40 antigen on human B cells, and which exhibit antagonist activity when bound to CD40 antigen on human B cells.
  • the anti-CD40 antibody is free of significant agonist activity in one B cell response.
  • the anti-CD40 antibody is free of significant agonist activity in assays of more than one B cell response (e.g., proliferation and differentiation, or proliferation, differentiation, and antibody production).
  • an anti-CD40 therapeutic agent e.g., an anti-CD40 antibody
  • an anti-CD40 therapeutic agent e.g., an anti-CD40 antibody
  • B cells proliferation assays B cell proliferation assays
  • Banchereau-Like-B cell proliferation assays T cell helper assays for antibody production
  • co-stimulation of B cell proliferation assays and assays for up-regulation of B cell activation markers.
  • Relevant assays are described in e.g., United States Patent No. 6,087,329 and the international patent applications published as WO 00/75348, WO 2005/044294, WO 2005/044304, WO 2005/044305,
  • the anti-CD40 antibody acts as an antagonist of one or more B cell responses.
  • the anti-CD40 antibody acts as an antagonist of at least one B cell response selected from the group consisting of B cell proliferation, B cell differentiation, antibody 5 production, intercellular adhesion, B cell memory generation, isotype switching, up- regulation of cell-surface expression of MHC Class II and CD80/86, and secretion of proinflammatory cytokines such as IL-8, IL-12, and TNF.
  • B cell proliferation B cell proliferation
  • B cell differentiation antibody 5 production
  • intercellular adhesion B cell memory generation
  • isotype switching up- regulation of cell-surface expression of MHC Class II and CD80/86
  • proinflammatory cytokines such as IL-8, IL-12, and TNF.
  • the anti-CD40 antibody may be an antagonist of B cell proliferation induced by soluble or cell-surface CD40L, as measured in a B cell proliferation assay. Suitable B cell proliferation assays are known in the art. Suitable B cell proliferation assays are also described below. In some embodiments, the antagonist anti-CD40 antibody stimulates B 15 cell proliferation at a level that is not more than about 25% greater than the B cell
  • a negative control i.e., at least 75% inhibition
  • a negative control i.e., at least 75% inhibition
  • the anti-CD40 antibody is an antagonist of B cell
  • the B cell proliferation induced by the other anti-CD40 antibody in the absence of the antagonist anti-CD40 antibody preferably not more than about 20%, 15%, 10%, 5%, 1%, 0.5%, or even not more than about 0.1% of the B cell proliferation induced by the other anti-CD40 antibody in the absence of the antagonist anti-CD40 antibody.
  • the anti-CD40 antibody is an antagonist of B cell proliferation that is induced by the cell line EL4B5 (Kwekkeboom et al. (1993)
  • the level of B cell proliferation stimulated by the EL4B5 cell line in the presence of the antagonist anti- CD40 antibody is not more than about 25% of the B cell proliferation induced by this cell line in the absence of the antagonist anti-CD40 antibody (i.e., at least 75% inhibition), preferably not more than about 20%, 15%, 10%, 5%, 1%, 0.5%, or even not more than about 0.1% of the B cell proliferation induced by this cell line in the absence of the antagonist anti-CD40 antibody.
  • the anti-CD40 antibody is an antagonist of human T- cell-induced antibody production by human B cells as measured in the human T-cell helper assay for antibody production by B cells.
  • the level of IgG antibody production, IgM antibody production, or both IgG and IgM antibody production by B cells stimulated by T cells in the presence of the antagonist anti-CD40 antibody is not more than about 50% of the respective antibody production by B cells stimulated by T cells in the absence of the antagonist anti-CD40 antibody (i.e., at least 75% inhibition), preferably not more than about 25%, 20%, 15%, 10%, 5%, 1%, 0.5%, or even not more than about 0.1% of the respective antibody production by B cells stimulated by T cells in the absence of the antagonist anti-CD40 antibody.
  • the following assays can be used to assess the antagonist activity of an anti-CD40 antibody.
  • Human B cells for these assays can be obtained, for example, by isolation from tonsils obtained from individuals undergoing tonsillectomies, essentially as described in De Groot et al. (1990) Lymphokine Research (1990) 9:321. Briefly, the tissue is dispersed with scalpel blades, phagocytic and NK cells are depleted by treatment with 5 mM L-leucine methyl ester and T cells are removed by one cycle of rosetting with sheep erythrocytes (SRBC) treated with 2-aminoethyl isothiouronium bromide.
  • SRBC sheep erythrocytes
  • the purity of the resulting B lymphocyte preparations can be checked by indirect immunofluorescent labelling with anti-(CD20) mAb B l (Coulter Clone, Hialeah, FA) or anti-(CD3) mAb O T3 (Ortho, Raritan, NJ) and a FITC -conjugated F(ab') 2 fragment of rabbit anti-(mouse Ig) (Zymed, San Francisco, CA), and FACS analysis.
  • B cells (4 x 10 4 per well) are cultured in 200 ⁇ IMDM supplemented with 10% fetal calf serum in flat bottom 96-well microtiter plates. B cells are stimulated by addition of immobilized anti-(IgM) antibodies (Immunobeads; 5 ⁇ g/ml; BioRad, Richmond, California). Where desired, 100 U/ml recombinant IL-2 is added. Varying concentrations of test monoclonal antibodies (mAbs) are added at the onset of the microcultures and proliferation is assessed at day 3 by measurement of the incorporation of ( 3 H)-thymidine after 18 hour pulsing. An antagonist anti-CD40 antibody does not significantly costimulate human B-cell proliferation in the presence of immobilized anti-IgM or in the presence of immobilized anti-IgM and IL-2.
  • immobilized anti-(IgM) antibodies Immunobeads; 5 ⁇ g/ml; BioRad, Richmond, California. Where
  • mouse 3T6 transfectant cells expressing the HR allellic form of human FcyRll are used.
  • B cells (2 x 10 4 per well) are cultured in flat-bottom microwelis in the presence of 1 x 10 4 transfectant cells (irradiated with 5000 Rad) in 200 ul IMDM supplemented with 10% fetal calf serum and 100 U/ml recombinant IL-4.
  • the 3T6 cells are allowed to adhere to the culture plastic for at least 5 hours.
  • Anti-CD40 mAbs are added at concentrations varying from 15 ng/ml to 2000 ng/ml and proliferation of B cells is assessed by measurement of thymidine incorporation at day 7, upon 18 hour pulsing with [ 3 H] thymidine.
  • Antagonist anti-CD40 monoclonal antibodies may also be characterized by their ability to inhibit stimulation of B-cell proliferation by an anti-CD40 antibody such as S2C6 (also known as SGN-14, which is reportedly an agonist of CD40 stimulation of proliferation of normal B cells; Francisco et al. (2000) Cancer Res.
  • An antagonist anti-CD40 antibody can inhibit the costimulation of anti-IgM induced human B-cell proliferation by mAb S2C6, for example, by at least 75% or more (i.e., S2C6-stimulated proliferation in the presence of an antagonist anti-CD40 antibody is no more than 25% of that observed in the absence of the antagonist anti-CD40 antibody).
  • S2C6-stimulated proliferation in the presence of an antagonist anti-CD40 antibody is no more than 25% of that observed in the absence of the antagonist anti-CD40 antibody.
  • no significant inhibition would be seen with equivalent amounts of non-relevant mAb 8E4, directed to ⁇ -glucocerebrosidase. Barneveld et al, supra. Such a result would indicate that the anti-CD40 mAbs does not deliver stimulatory signals for the
  • PMA phorbol-12-myristate 13 -acetate
  • B cells (1000 per well) are cultured together with irradiated (5000 Rad) EL4B5 cells (5 x 10 4 per well) in flat bottom microtiter plates in 200 ⁇ IMDM supplemented with 10% heat-inactivated fetal calf serum, 5 ng/ml phorbol-I2-myristate 13-acetate and 5% human T-cell supernatant.
  • mAbs are added at varying concentrations at the onset of the cultures and thymidine incorporation is assessed at day 6 after 18 hour pulsing with [ 3 H]-thymidine.
  • T-cell supernatant For the preparation of T-cell supernatant, purified T cells are cultured at a density of 10 6 /ml for 36 hours in the presence of 1 ⁇ ⁇ 1 PHA and 10 ng/ml PMA. Wen et al. (1987) Eur. J. Immunol. (1987) 17:887. T-cell supernatant is obtained by centrifugation of the cells and stored at -20°C. The effectiveness of T-cell supernatants in enhancing proliferation of human B cells in EL4B5-B cell cultures is tested and the most effective supernatants are pooled for use in experiments.
  • a monoclonal antibody such as MOPC-141 (IgG2b) can be added as a control.
  • MOPC-141 IgG2b
  • An antagonist anti-CD40 antibody may function as an antagonist of antibody production by B cells.
  • An anti-CD40 antibody can be tested for this type of antagonist activity by assessing the antibody's ability to inhibit antibody production by B cells that have been stimulated in a contact-dependent manner with activated T cells in a T cell helper assay. In this manner, 96-well tissue culture plates are coated with a 1 :500 dilution of ascites fluid of anti-CD3 mAb CLB-T3/3 (CLB, Amsterdam, The
  • costimulatory mAbs are added: anti CD2 mAbs CLB-Tl 1.1/1 and CLB-Tl 1.2/1 (CLB, Amsterdam, The Netherlands), both ascites 1 : 1000 and anti- CD28 mAb CLB-28/1 (CLB, Amsterdam, The Netherlands).
  • tonsillar T cells irradiated, 3000 Rad; 10 5 per well
  • tonsillar B cells 10 4 per well
  • rIL-2 (20 U/ml
  • human tonsillar B cells ( 10 4 /well) are cultured together with irradiated purified T cells (3000 rad, 10 5 /well) in 96-well plates, coated with anti-CD3 mAb and with or without different mAbs to costimulate the T cells. After 8 days of culture the supernatants are harvested for the determinatio of antibody production by the B cells. Antibody production by the B cells is assessed by the ELISA assay described below. The anti-CD40 antibody of interest is added in varying concentrations from the onset of the cultures. As a control, mAb MOPC-141 can be added.
  • An antagonist anti-CD40 antibody can inhibit IgG and IgM antibody production of B cells stimulated by human T cells by at least 50% or more (i.e., T cell-induced antibody production by B cells in the presence of an antagonist anti-CD40 antibody is no more than 50% of that observed in the absence of the antagonist anti-CD40 antibody).
  • a control antibody such as MOPC-141 would have no significant effect on T cell-induced antibody production by B cells.
  • the concentrations of human IgM and IgG are estimated by ELISA.
  • 96-well ELISA plates are coated with 4 ⁇ g/ml mouse anti-human IgG mAb MH 16-01 (CLB, Amsterdam, The Netherlands) or with 1.2 ⁇ g/ml mouse anti-human IgM mAb 4102
  • Antagonist anti-CD40 antibodies are known in the art. See, for example, the human anti-CD40 antibody produced by the hybridoma designated F4-465 disclosed in U.S. Patent Application Publication Nos. 20020142358 and 20030059427. F4-465 was obtained from the HAC mouse (Kuroiwa et al. (2000) Nature Biotech. 10: 1086 (2000)) and therefore expresses the human lambda light chain.
  • the anti-CD40 antibody for use in the methods of the present invention will preferably have another mechanism of action against a target cell.
  • the anti-CD40 antibody will preferably have ADCC activity.
  • anti-CD40 antibodies that share the binding characteristics of HCD122 (produced by the hybridoma cell line deposited with the ATCC (American Type Culture Collection; 10801 University Boulevard., Manassas, Virginia 20110-2209 (USA)) on September 17, 2003, as Patent Deposit No. PTA-5543).
  • Such antibodies include, but are not limited to:
  • an antibody comprising an amino acid sequence selected from the group consisting of the sequence shown in SEQ ID NO:2, the sequence shown in SEQ ID NO:4, the sequence shown in SEQ ID NO:5, both the sequences shown in SEQ ID NO:2 and SEQ ID NO:4, and both the sequences shown in SEQ ID NO:2 and SEQ ID NO:5;
  • an antibody comprising an amino acid sequence selected from the group consisting of the sequence shown in SEQ ID NO: 17, the sequence shown in SEQ ID NO: 19, the sequence shown in SEQ ID NO:20, both the sequences shown in SEQ ID NO: 17 and SEQ ID NO: 19, and both the sequences shown in SEQ ID NO: 17 and SEQ ID NO:20;
  • an antibody comprising an amino acid sequence selected from the group consisting of the sequence shown in SEQ ID NO: 16, the sequence shown in SEQ ID NO: 18, and both the sequences shown in SEQ ID NO: 16 and SEQ ID NO: 18;
  • an antibody having an amino acid sequence encoded by a nucleic acid molecule comprising a nucleotide sequence selected from the group consisting of the sequence shown in SEQ ID NO: 1 , the sequence shown in SEQ ID NO:3, and both the sequences shown in SEQ ID NO:l and SEQ ID NO:3;
  • V L light chain variable domain
  • V H heavy chain variable domain
  • V L light chain variable domain
  • V H heavy chain variable domain
  • an antibody comprising a light chain variable domain containing the complementarity determining region (CDR) residues of SEQ ID NO:2;
  • an antibody comprising a light chain variable domain containing the complementarity determing region (CDR) residues of SEQ ID NO:2 and a heavy chain variable domain containing the complementarity determining region (CDR) residues of SEQ ID NO:4;
  • An anti-CD40 antibody obtained from a CHO cell containing one or more expression vectors encoding the antibody can be used in the methods of the invention.
  • the monoclonal antibody HCD122 produced by the hybridoma cell line deposited with the ATCC as Patent Deposit No. PTA-5543, is particularly preferred for use in the methods, compositions, uses, and kits of the invention.
  • the monoclonal antibody HCD122 binds domain 2 of human CD40 antigen, whereas earlier anti-CD40 antibodies having antagonistic properties were found to bind to other domains of human CD40.
  • the HCD 122 monoclonal antibody binds soluble CD40 in ELISA-type assays, prevents the binding of CD40-ligand to cell-surface CD40, and displaces the pre-bound CD40-ligand, as determined by flow cytometric assays.
  • HCD122 acts as antagonist anti- CD40 antibody.
  • HCD122 does not induce strong proliferation of human lymphocytes from normal subjects.
  • the antibody is able to; kill CD40-expressing target cells by antibody dependent cellular cytotoxicity (ADCC).
  • the binding affinity of HCD122 for human CD40 is 5xl0 ⁇ 10 M, as determined by the BiacoreTM assay.
  • the nucleotide and amino acid sequences of the HCD122 antibody are known (e.g., see WO 2005/044854). Further, the mouse hybridoma line 153.8E2.D10.D6.12.12 (CMCC# 12056), which expresses the HCD122 antibody, has been deposited with the American Type Culture Collection [ATCC; 10801 University Boulevard., Manassas, Virginia 201 10-2209 (USA)] on September 17, 2003, under Patent Deposit Number PTA-5543.
  • ATCC American Type Culture Collection
  • the complete sequence for the light chain of HCD122 is set forth in SEQ ID NO:2, which includes the leader sequence (residues 1 -20 of SEQ ID NO:2), the variable region (residues 21-132 of SEQ ID NO:2), and the constant region (residues 133-239 of SEQ ID NO:2).
  • the complete sequence for the heavy chain of HCD122 is set forth in SEQ ID NO:4, which includes the leader sequence (residues 1-19 of SEQ ID NO:4), the variable region (residues 20-139 of SEQ ID NO:4), and the constant regions (residues 140-469 of SEQ ID NO:4).
  • HCD122 The complete sequence for a variant of HCD122 is set forth in SEQ ID NO:5, which includes the leader sequence (residues 1-19 of SEQ ID NO:5), the variable region (residues 20-139 of SEQ ID NO:5), and the constant regions (residues 140-469 of SEQ ID NO: 5).
  • This variant differs from HCD122 in that it contains a substitution of a serine residue for the alanine residue at position 153 of SEQ ID NO:4, which is within the constant region.
  • the nucleotide sequences encoding the light and heavy chains of HCD122 are set forth in SEQ ID NO: 1 (coding sequence for the light chain of HCD122) and SEQ ID NO:3 (coding sequence for the heavy chain of HCD122).
  • the amino acid sequence for the variable region of the HCD122 light chain without the leader sequence i.e., residues 21 -132 of SEQ ID NO:2
  • the amino acid sequence for the variable and constant regions of the HCD122 light chain without the leader sequence i.e., residues 21-239 of SEQ ID NO:2 is set forth in SEQ ID NO:17.
  • the amino acid sequence for the variable region of the HCD122 heavy chain without the leader sequence is set forth in SEQ ID NO: 18.
  • the amino acid sequence for the variable and constant regions of the HCD122 heavy chain without the leader sequence is set forth in SEQ ID NO: 19.
  • the amino acid sequence for the variable and constant regions of the HCD122 heavy chain variant is set forth in SEQ ID NO:20.
  • Anti-CD40 antibodies for use in the methods, compositions, uses, and kits of the present invention include antibodies differing from the HCD122 monoclonal antibody but retaining the CDRs, and antibodies with one or more amino acid addifion(s), deletion(s), or substitution(s).
  • HCD 122 is a fully human antibody, but can be further de-immunized if desired.
  • De-immunized anti-CD40 antibodies can be produced using known methods, e.g., as described in WO 98/52976 and WO 00/34317. In this manner, residues within the anti-CD40 antibodies may be modified so as to render the antibodies less immunogenic to humans while retaining their therapeutic activity.
  • Any known antibody having the binding specificity of interest can have sequence variations produced using methods described in, for example, EP 0983303,
  • WO 00/34317 and WO 98/52976.
  • sequences within the CDR can cause an antibody to bind to MHC Class II and trigger an unwanted helper T-cell response in certain patients.
  • a conservative substitution can allow the antibody to retain binding activity yet lose its ability to trigger an unwanted T-cell response.
  • Any such conservative or non-conservative substitutions can be made using art-recognized methods, such as those noted elsewhere herein, and the resulting antibodies can also be used in the methods of the present invention.
  • the variant antibodies can be routinely tested for the particular activity, for example, antagonist activity, affinity, and specificity using methods described herein.
  • amino acid sequence variants of an antagonist anti-CD40 antibody can be prepared by mutations in the cloned DNA sequence encoding the antibody of interest.
  • Methods for mutagenesis and nucleotide sequence alterations are well known in the art. See, for example, Walker and Gaastra, eds. (1983) Techniques in Molecular Biology (MacMillan Publishing Company, New York); Kunkel (1985) Proc. Natl. Acad. Sci. USA 82:488-492; Kunkel et al. (1987) Methods Enzymol. 154:367-382; Sambrook et al. (1989) Molecular Cloning: A
  • variants of an antibody of interest for example, an antagonist anti- CD40 antibody polypeptide of interest
  • modifications may be made such that variants continue to possess the desired activity, /. e. , similar binding affinity and, in the case of antagonist anti-CD40 antibodies, are capable of specifically binding to a human CD40 antigen expressed on the surface of a human cell, and being free of significant agonist activity but exhibiting antagonist activity when bound to a CD40 antigen on a human CD40-expressing cell.
  • any mutations made in the DNA encoding the variant polypeptide must not place the sequence out of reading frame and preferably will not create complementary regions that could produce secondary mRNA structure (e.g., see EP 0075444).
  • constant region of an antibody for example, an antagonist anti- CD40 antibody
  • an antibody can be mutated to alter effector function in a number of ways. For example, see U.S. Patent 6,737,056 Bl and U.S. Patent Application Publication No. 2004/0132101A1, which disclose Fc mutations that optimize antibody binding to Fc receptors.
  • variants of a reference antibody for example, an antagonist anti-CD40 antibody
  • percent sequence identity is determined using the Smith- Waterman homology search algorithm using an affine gap search with a gap open penalty of 12 and a gap extension penalty of 2, BLOSUM matrix of 62.
  • a variant may, for example, differ from the reference antibody, for example, an antagonist anti-CD40 antibody, by as few as 1 to 15 amino acid residues, as few as 1 to 10 amino acid residues, such as 6-10, as few as 5, as few as 4, 3, 2, or even 1 amino acid residue.
  • the contiguous segment of the variant amino acid sequence may have additional amino acid residues or deleted amino acid residues with respect to the reference amino acid sequence.
  • the contiguous segment used for comparison to the reference amino acid sequence will include at least 20 contiguous amino acid residues, and may be 30, 40, 50, or more amino acid residues. Corrections for sequence identity associated with conservative residue substitutions or gaps can be made (see Smith- Waterman homology search algorithm).
  • an antibody capable of specifically binding CD40 and retaining antagonist activity depends on a number of factors.
  • ionizable amino and carboxyl groups are present in an antibody molecule, a particular polypeptide may be obtained as an acidic or basic salt, or in neutral form. All such preparations that retain their biological activity when placed in suitable environmental conditions are included in the definition of antagonist anti-CD40 antibodies as used herein.
  • the primary amino acid sequence of the polypeptide may be augmented by derivatization using sugar moieties (glycosylation) or by other supplementary molecules such as lipids, phosphate, acetyl groups and the like. It may also be augmented by conjugation with saccharides.
  • the art provides substantial guidance regarding the preparation and use of antibody variants.
  • preparing the anti-CD40 antibody variants one of skill in the art can readily determine which modifications to the native protein nucleotide or amino acid sequence will result in a variant that is suitable for use as a therapeutically active component of a pharmaceutical composition used in the methods of the present invention.
  • the anti-CD40 antibody for use in the methods of the invention preferably possesses at least one of the following biological activities in vitro and/or in vivo:
  • Immunol. 169:5401- 5404 cytotoxicity via any mechanism (including, but not limited to, antibody-dependent cell-mediated cytotoxicity (ADCC), complement-dependent cytotoxicity (CDC), down- regulation of proliferation, and/or apoptosis in target cells), modulation of target cell cytokine secretion and/or cell surface molecule expression, and combinations thereof.
  • Assays for such biological activities can be performed as described herein. See also the assays described in Schultze et al. (1998) Proc. Natl. Acad. Sci. USA 92:8200-8204; Denton et al. (1998) Pediatr. Transplant. 2:6-15; Evans et al. (2000) J. Immunol.
  • an antibody it is possible to engineer an antibody to have increased ADCC activity.
  • the carboxy-terminal half of the CH2 domain is important for ADCC mediated through the FcRIII receptor. Since the CH2 and hinge regions have an important role in effector functions, a series of multiple-domain antibodies that contain extra CH2 and/or hinge regions may be created and investigated for any changes in effector potency (see Greenwood et al. (1994) Ther. Immunol. l(5):247-55).
  • An alternative approach may be to engineer extra domains in parallel, for example, through creation of dimers by engineering a cysteine into the H-chain of a chimeric Ig (see Shopes (1992) J. Immunol. 148(9):2918-2922).
  • changes to increase ADCC activity may be engineered by introducing mutations into the Fc region (see, for example, U.S. Patent No. 6,737,056 Bl), expressing cells in fucosyl transferase deficient cell lines (see, for example, U.S. Patent Application Publication No. 2003/0115614), or effecting other changes to antibody glycosylation (see, for example, U.S. Patent No. 6,602,684).
  • CD40-antigen epitopes identified herein is a "competitive binding assay".
  • Competitive binding assays are serological assays in which unknowns are detected and quantitated by their ability to inhibit the binding of a labeled known ligand to its specific antibody. This is also referred to as a competitive inhibition assay.
  • labeled CD40 polypeptide is precipitated by candidate antibodies in a sample, for example, in combination with monoclonal antibodies raised against one or more epitopes of anti-CD40 monoclonal antibodies.
  • Anti-CD40 antibodies that specifically react with an epitope of interest can be identified by screening a series of antibodies prepared against a CD40 protein or fragment of the protein comprising the particular epitope of the CD40 protein of interest.
  • epitopes of interest include epitopes comprising linear and/or nonlinear amino acid residues of the short isoform of human CD40 (see GenBank Accession No. NP_690593) set forth in SEQ ID NO:7, encoded by the sequence set forth SEQ ID NO:6; see also GenBank Accession No. NM_152854), or of the long isoform of human CD40 (see GenBank Accession Nos. CAA43045 and NP_001241, set forth in SEQ ID NO:9, encoded by the sequence set forth in SEQ ID NO:8; see GenBank Accession Nos.
  • Antibodies employed in such immunoassays may be labeled or unlabeled.
  • Unlabeled antibodies may be employed in agglutination; labeled antibodies may be employed in a wide variety of assays, employing a wide variety of labels. Detection of the formation of an antibody-antigen complex between an anti-CD40 antibody and an epitope of interest can be facilitated by attaching a detectable substance to the antibody. Suitable detection means include the use of labels such as radionuclides, enzymes, coenzymes, fluorescers, chemiluminescers, chromogens, enzyme substrates or co-factors, enzyme inhibitors, prosthetic group complexes, free radicals, particles, dyes, and the like.
  • labels such as radionuclides, enzymes, coenzymes, fluorescers, chemiluminescers, chromogens, enzyme substrates or co-factors, enzyme inhibitors, prosthetic group complexes, free radicals, particles, dyes, and the like.
  • suitable enzymes include horseradish peroxidase, alkaline phosphatase, ⁇ - galactosidase, or acetylcholinesterase;
  • suitable prosthetic group complexes include streptavidin biotin and avidin/biotin;
  • suitable fluorescent materials include umbelliferone, fluorescein, fluorescein isothiocyanate, rhodamine,
  • labeled reagents may be used in a variety of well-known assays, such as radioimmunoassays, enzyme immunoassays, e.g., ELISA, fluorescent immunoassays, and the like. See for example, U.S. Patent Nos. 3,766,162; 3,791 ,932; 3,817,837; and 4,233,402.
  • the combination therapy of the invention addresses problems associated with known therapies for diseases or conditions associated with neoplastic B- cell growth, including therapy using rituximab (commercially available under the tradename Rituxan®).
  • Rituximab has been shown to be an effective treatment for low-, intermediate-, and high-grade non-Hodgkin's lymphoma (NHL) and active in other B-cell malignancies (see for example, Maloney et al. (1994) Blood 84:2457-2466), McLaughlin et al. (1998) J. Clin. Oncol. 16:2825-2833, Maloney et al. (1997) Blood 90:2188-2195, Hainsworth et al.
  • NHL non-Hodgkin's lymphoma
  • Rituximab is licensed for treatment of relapsed B cell low-grade or follicular non-Hodgkin's lymphoma (NHL). In follicular lymphoma, which is the most common indolent non-Hodgkin lymphoma (NHL), rituximab combined with
  • patients tend to become refractory to rituximab over time (see Witzig et al. (2002) J. Clin. Oncol. 20:3262, Grillo- Lopez et al. (1998) J. Clin. Oncol. 16:2825, or Jazirehi et al. (2003) Mol. Cancer Then 2: 1183-1193).
  • some patients lose CD20 expression on malignant B cells after anti-CD20 antibody therapy (Davis et al. (1999) Clin. Cancer Res. 5:611).
  • the invention uses combination therapy with bendamustine and anti-CD40 antibodies for treating diseases or conditions associated with neoplastic B-cell growth.
  • Bendamustine (TreandaTM; Cephalon, Inc., Frazer, Pa) is a novel alkylator whose mechanisms of action involve induction of apoptosis through activation of DNA-damage stress responses, inhibition of mitotic checkpoints, and induction of mitotic catastrophe (Leoni et al. (2008) Clin Cancer Res. 2008;14:309-317).
  • the compound also contains a benzimidazole ring, which may confer purine analogue-like properties in addition to the alkylating properties.
  • bendamustine is administered at therapeutically effective doses ranging between about 60 mg/m 2 and about 120 mg m 2 , including, for example, 60 mg/m 2 , 65 mg/m 2 , 70 mg/m 2 , 75 mg/m 2 , 80 mg/m 2 , 85 mg/m 2 , 90 mg/m 2 , 95 mg/m 2 , 100 mg/m 2 , 105 mg m 2 , 110 mg/m 2 , 115 mg/m 2 , and 120 mg/m 2 , and other such doses falling within the range of about 60 mg/m 2 and 120 mg/m 2 .
  • bendamustine In order to reduce hematologic toxicity, dose modifications, and treatment delays, bendamustine preferably is administered at a dose of about about 85 mg/m 2 , about 90 mg/m 2 , or about 95 mg/m 2 on Days 1 and 2 of any given cycle of combination therapy with the anti-CD40 antibody (such as HCD 122).
  • the bendamustine is administered intravenously (iv) by infusion over 60 minutes according to the most up-to-date prescription guidelines or other applicable institutional or regional guidelines.
  • the anti-CD40 antibodies of this invention are administered at a concentration that is therapeutically effective to treat a disease or condition associated with neoplastic CD40-expressing B cells.
  • the antibodies may be formulated using a variety of acceptable carrier and/or excipients known in the art.
  • the anti-CD40 antibody may be administered by a parenteral route of administration. Typically, the antibodies are administered by injection, either intravenously or subcutaneously. Methods to accomplish this administration are known to those of ordinary skill in the art.
  • Intravenous administration occurs preferably by infusion over a period of about less than 1 hour to about 10 hours (more preferably less than 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 hours). Subsequent infusions may be administered over a period of about less than 1 to about 6 hours, including, for example, about 1 to about 4 hours, about 1 to about 3 hours, or about 1 to about 2 hours. In some embodiments, the anti-CD40 antibody is administered by infusion over a period of about 2 to about 6 hours. Alternatively, a dose can be admininstered subcutaneously.
  • a pharmaceutical composition of the invention is formulated to be compatible with its intended route of administration.
  • Solutions or suspensions used for parenteral application can include the following components: a sterile diluent such as water for injection, saline solution; antibacterial agents such as benzyl alcohol or methyl parabens; antioxidants such as ascorbic acid or sodium bisulfite; chelating agents such as ethylenediaminetetraacetic acid; buffers such as acetates, citrates or phosphates and agents for the adjustment of tonicity such as sodium chloride or dextrose. pH can be adjusted with acids or bases, such as hydrochloric acid or sodium hydroxide.
  • the parenteral preparation can be enclosed in ampoules, disposable syringes, or multiple dose vials made of glass or plastic.
  • the anti-CD40 antibodies are typically provided b standard technique within a pharmaceutically acceptable buffer, for example, sterile saline, sterile buffered water, combinations of the foregoing, etc.
  • a pharmaceutically acceptable buffer for example, sterile saline, sterile buffered water, combinations of the foregoing, etc.
  • Methods for preparing parenterally administrable agents are described in Remington: The Science and Practice of Pharmacy (21 st edition, Lippincott Williams & Wilkins, May 2005). See also, for example, WO 98/56418, which describes stabilized antibody pharmaceutical formulations suitable for use in the methods of the present invention.
  • the amount of anti-CD40 antibody to be administered is readily determined by one of ordinary skill in the art in view of the guidance provided herein. Factors influencing the mode of administration and the respective amount of anti-CD40 antibody include, but are not limited to, the the severity of the disease, the history of the disease, and the age, height, weight, health, type of disease, and physical condition of the individual undergoing therapy or response to antibody infusion. Similarly, the amount of anti-CD40 antibody to be administered will be dependent upon the mode of
  • the amount to be administered may be in the range from about 0.1 mg/kg to about 35 mg/kg, from about 0.5 mg/kg to about 30 mg/kg, from about 1 mg/kg to about 30 mg/kg, from about 3 mg/kg to about 30 mg/kg, from about 3 mg/kg to about 25 mg/kg, from about 3 mg/kg to about 20 mg/kg, or from about 5 mg/kg to about 15 mg/kg.
  • the dose can be 0.3 mg/kg, 0.5 mg/kg, 1 mg/kg, 1.5 mg/kg, 2 mg/kg, 2.5 mg/kg, 3 mg/kg, 3.5 mg/kg, 4 mg/kg, 4.5 mg/kg, 5 mg/kg, 5.5 mg/kg, 6 mg/kg, 6.5 mg/kg, 7 mg/kg, 7.5 mg/kg, 8 mg/kg, 8.5 mg/kg, 9 mg/kg, 9.5 mg/kg, 10 mg/kg, 15 mg/kg, 20 mg/kg, 25 mg/kg, 30 mg/kg, or 35 mg/kg, or other such doses falling within the range of about 0.3 mg/kg to about 35 mg/kg.
  • Treatment of a subject with a therapeutically effective amount of an antibody can include a single treatment or, preferably, can include a series of treatments.
  • the methods of the invention may comprise administration of multiple doses of anti-CD40 antibody.
  • the method may comprise administration of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, or more therapeutically effective separate doses of a pharmaceutical composition comprising an anti-CD40 antibody.
  • the frequency and duration of administration of multiple doses of the pharmaceutical compositions comprising anti- CD40 antibody can be readily determined by one of skill in the art without undue experimentation when equipped with the disclosure provided herein.
  • the same therapeutically effective dose of an anti-CD40 antibody can be administered over the course of a treatment period.
  • different therapeutically effective doses of an anti-CD40 antibody can be used over the course of a treatment period.
  • a subject is treated with anti-CD40 antibody in the range of between about 0.1 to 20 mg/kg body weight, once per week for between about 1 to 10 weeks, preferably between about 2 to 8 weeks, more preferably between about 3 to 7 weeks, and even more preferably for about 4, 5, or 6 weeks. Treatment may occur at intervals of every 2 to 12 months to prevent relapse or upon indication of relapse. It will also be appreciated that the effective dosage of antibody used for treatment may increase or decrease over the course of a particular treatment. Changes in dosage may result and become apparent from the results of diagnostic assays.
  • the dosing regimen includes a first administration of a therapeutically effective dose of at least one anti-CD40 antibody on days 1 , 8, 15, and 22 of a treatment period.
  • the dosing regimen includes a dosing regimen having a first administration of a therapeutically effective dose of at least one anti-CD40 antibody daily, or on days 1, 3, 5, and 7 of a week in a treatment period; a dosing regimen including a first administration of a therapeutically effective dose of at least one anti- CD40 antibody on days 1 and 3-4 of a week in a treatment period; and a dosing regimen including a first administration of a therapeutically effective dose of at least one anti- CD40 antibody on day 1 of a week in a treatment period.
  • the treatment period may comprise at least 1 week, at least 2 weeks, at least 3 weeks, at least a month, at least 2 months, at least 3 months, at least 6 months, or at least a year. Treatment periods may be subsequent or separated from each other by at least a week, at least 2 weeks, at least a month, at least 3 months, at least 6 months, or at least a year.
  • the initial therapeutically effective dose of an anti-CD40 antibody as defined elsewhere herein can be in the lower dosing range (i.e. , about 0.3 mg/kg to about 20 mg kg) with subsequent doses falling within the higher dosing range (i.e. , from about 20 mg/kg to about 50 mg/kg).
  • CD40 antibody as defined elsewhere herein can be in the upper dosing range (i.e., about 20 mg/kg to about 50 mg/kg) with subsequent doses falling within the lower dosing range (i.e., 0.3 mg/kg to about 20 mg/kg).
  • anti- CD40 antibody therapy may be initiated by administering a "loading dose" of the antibody to the subject in need therapy.
  • loading dose is intended an initial dose of the anti-CD40 antibody that is administered to the subject, where the dose of the antibody administered falls within the higher dosing range (i.e. , from about 20 mg/kg to about 50 mg/kg).
  • the "loading dose” can be administered as a single administration, for example, a single infusion where the antibody is administered IV, or as multiple administrations, for example, multiple infusions where the antibody is administered IV, so long as the complete "loading dose” is administered within about a 24-hour period.
  • the subject is then administered one or more additional therapeutically effective doses of the anti-CD40 antibody.
  • Subsequent therapeutically effective doses can be administered, for example, according to a weekly dosing schedule, or once every two weeks, once every three weeks, or once every four weeks. In such embodiments, the subsequent therapeutically effective doses generally fall within the lower dosing range (i.e., 0.3 mg/kg to about 20 mg kg).
  • the subsequent therapeutically effective doses of the anti-CD40 antibody are administered according to a "maintenance schedule", wherein the therapeutically effective dose of the antibody is administered once a month, once every 6 weeks, once every two months, once every 10 weeks, once every three months, once every 14 weeks, once every four months, once eveiy 18 weeks, once every five months, once every 22 weeks, once every six months, once every 7 months, once every 8 months, once every 9 months, once every 10 months, once every 1 1 months, or once every 12 months.
  • the therapeutically effective doses of the anti-CD40 antibody fall within the lower dosing range (i.e., 0.3 mg/kg to about 20 mg/kg), particularly when the subsequent doses are administered at more frequent intervals, for example, once every two weeks to once every month, or within the higher dosing range (i.e., from about 20 mg/kg to about 50 mg/kg), particularly when the subsequent doses are administered at less frequent intervals, for example, where subsequent doses are administered about one month to about 12 months apart.
  • the anti-CD40 antibodies present in the pharmaceutical compositions described herein for use in the methods of the invention may be native or obtained by recombinant techniques, and may be from any source, including mammalian sources such as, e.g., mouse, rat, rabbit, primate, pig, and human.
  • mammalian sources such as, e.g., mouse, rat, rabbit, primate, pig, and human.
  • polypeptides are derived from a human source, and more preferably are recombinant, human proteins from hybridoma cell lines.
  • compositions comprising an anti-CD40 antibody having the binding properties described herein as the therapeutically active component can be used in the methods of the invention.
  • liquid, lyophilized, or spray-dried compositions comprising one or more of the anti-CD40 antibodies may be prepared as an aqueous or nonaqueous solution or suspension for subsequent administration to a subject in accordance with the methods of the invention.
  • Each of these compositions will comprise at least one anti-CD40 antibody as a therapeutically or prophylactically active component.
  • therapeutically or prophylactically active component is intended the anti-CD40 antibody is specifically incorporated into the composition to bring about a desired therapeutic or prophylactic response with regard to treatment, prevention, or diagnosis of a disease or condition within a subject when the pharmaceutical composition is administered to that subject.
  • the pharmaceutical compositions comprise appropriate stabilizing agents, bulking agents, or both to minimize problems associated with loss of protein stability and biological activity during preparation and storage.
  • Formulants may be added to pharmaceutical compositions comprising an anti- CD40 antibody.
  • These formulants may include, but are not limited to, oils, polymers, vitamins, carbohydrates, amine acids, salts, buffers, albumin, surfactants, or bulking agents.
  • carbohydrates include sugar or sugar alcohols such as mono-, di-, or polysaccharides, or water soluble glucans.
  • the saccharides or glucans can include fructose, glucose, trehalose, mannose, sorbose, xylose, maltose, sucrose, dextran, pullulan, dextrin, a- and ⁇ -cyclodextrin, soluble starch, hydroxy ethyl starch, and carboxymethylcellulose, or mixtures thereof.
  • "Sugar alcohol” is defined as a C4 to Cg hydrocarbon having a hydroxyl group 'and includes galactitol, inositol, mannitol, xylitol, sorbitol, glycerol, and arabitol. These sugars or sugar alcohols may be used individually or in combination.
  • the sugar or sugar alcohol concentration is between 1.0% and 7% w/v., more preferably between 2.0% and 6.0% w/v.
  • amino acids include levorotary (L) forms of carnitine, arginine, and betaine; however, other amino acids may be added.
  • Preferred polymers include polyvinylpyrrolidone (PVP) with an average molecular weight between 2,000 and 3,000, or polyethylene glycol (PEG) with an average molecular weight between 3,000 and 5,000.
  • PVP polyvinylpyrrolidone
  • PEG polyethylene glycol
  • Surfactants that can be added to the formulation are shown in EP Nos. 270,799 and 268,1 10.
  • the formulants to be incorporated into a pharmaceutical composition should provide for the stability of the anti-CD40 antibody. That is, the anti-CD40 antibody should retain its physical and/or chemical stability and have the desired biological activity, i.e., one or more of the antagonist activities defined herein above.
  • a stable antibody pharmaceutical formulation provides for stability of the anti-CD40 antibody when stored at room temperature (about 25°C) for at least 1 month, at least 3 months, or at least 6 months, and/or is stable at about 2-8 °C for at least 6 months, at least 9 months, at least 12 months, at least 18 months, at least 24 months.
  • a protein such as an antibody when formulated in a pharmaceutical composition, is considered to retain its physical stability at a given point in time if it shows no visual signs (i.e. , discoloration or loss of clarity) or measurable signs (for example, using size- exclusion chromatography (SEC) or UV light scattering) of precipitation, aggregation, and/or denaturation in that pharmaceutical composition.
  • SEC size- exclusion chromatography
  • UV light scattering for example, using size- exclusion chromatography (SEC) or UV light scattering
  • composition is considered to retain its chemical stability at a given point in time if measurements of chemical stability are indicative that the protein (i.e., antibody) retains the biological activity of interest in that pharmaceutical composition.
  • Methods for monitoring changes in chemical stability include, but are not limited to, methods to detect chemically altered forms of the protein such as result from clipping, using, for example, SDS-PAGE, SEC, and/or matrix-assisted laser desorption ionization/time of flight mass spectrometry; and; degradation associated with changes in molecular charge (for example, associated with deamidation), using, for example, ion-exchange chromatography. See, for example, the methods disclosed herein below.
  • An anti-CD40 antibody when formulated in a pharmaceutical composition, is considered to retain a desired biological activity at a given point in time if the desired biological activity at that time is within about 30%, preferably within about 20% of the desired biological activity exhibited at the time the pharmaceutical composition was prepared as determined in a suitable assay for the desired biological activity.
  • Assays for measuring the desired biological activity of the anti-CD40 antibodies can be performed as described in the Examples herein. See also the assays described in Schultze et al. (1998) Proc. Natl. Acad. Sci. USA 92:8200-8204; Denton et al. (1998) Pediatr. Transplant. 2:6- 15; Evans et al. (2000) J. Immunol.
  • the anti-CD40 antibody is formulated in a liquid pharmaceutical formulation.
  • the anti-CD40 antibody can be prepared using any method known in the art, including those methods disclosed herein above.
  • the anti-CD40 antibody may be recombinantly produced in a CHO cell line.
  • the anti-CD40 antibody is to be stored prior to its formulation, it can be frozen, for example, at ⁇ -20°C, and then thawed at room temperature for further formulation.
  • the liquid pharmaceutical formulation comprises a therapeutically effective amount of the anti-CD40 antibody.
  • the amount of antibody thereof present in the formulation takes into consideration the route of administration and desired dose volume.
  • the liquid pharmaceutical composition comprises the anti-CD40 antibody at a concentration of about 0.1 mg/ml to about 50.0 mg/ml, about 0.5 mg/ml to about 40.0 mg/ml, about 1.0 mg/ml to about 30.0 mg/ml, about 5.0 mg/ml to about 25.0 mg/ml, about 5.0 mg/ml to about 20.0 mg/ml, or about 15.0 mg/ml to about 25.0 mg/ml.
  • the liquid pharmaceutical composition comprises the anti-CD40 antibody at a concentration of about 0.1 mg/ml to about 5.0 mg/ml, about 5.0 mg/ml to about 10.0 mg/ml, about 10.0 mg/ml to about 15.0 mg/ml, about 15.0 mg/ml to about 20.0 mg/ml, about 20.0 mg/ml to about 25.0 mg/ml, about 25.0 mg ml to about 30.0 mg/ml, about 30.0 mg/ml to about 35.0 mg/ml, about 35.0 mg/ml to about 40.0 mg/ml, about 40.0 mg/ml to about 45.0 mg/ml, or about 45.0 mg/ml to about 50.0 mg/ml.
  • the liquid pharmaceutical composition comprises the anti-CD40 antibody at a concentration of about 15.0 mg/ml, about 16.0 mg/ml, about 17.0 mg/ml, about 18.0 mg/ml, about 19.0 mg/ml, about 20.0 mg/ml, about 21.0 mg/ml, about 22.0 mg/ml, about 23.0 mg/ml, about 24.0 mg/ml, or about 25.0 mg/ml.
  • the liquid pharmaceutical composition comprises the anti-CD40 antibody and a buffer that maintains the pH of the formulation in the range of about pH 5.0 to about pH 7.0, including about pH 5.0, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6.0, 6.1 , 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, 7.0.
  • the buffer maintains the pH of the formulation in the range of about pH 5.0 to about pH 6.5, about pH 5.0 to about pH 6.0, about pH 5.0 to about pH 5.5, about pH 5.5 to about 7.0, about pH 5.5 to about pH 6.5, or about pH 5.5 to about pH 6.0.
  • Suitable buffer that maintains the pH of the liquid anti-CD40 antibody formulation in the range of about pH 5.0 to about pH 7.0 can be used in the formulation, so long as the physicochemical stability and desired biological activity of the antibody are retained as noted herein above.
  • Suitable buffers include, but are not limited to, conventional acids and salts thereof, where the counter ion can be, for example, sodium, potassium, ammonium, calcium, or magnesium.
  • Examples of conventional acids and salts thereof that can be used to buffer the pharmaceutical liquid formulation include, but are not limited to, succinic acid or succinate, citric acid or citrate, acetic acid or acetate, tartaric acid or tartarate, phosphoric acid or phosphate, gluconic acid or gluconate, glutamic acid or glutamate, aspartic acid or aspartate, maleic acid or maleate, and malic acid or malate buffers.
  • the buffer concentration within the formulation can be from about 1 mM to about 50 niM, including about 1 mM, 2 mM, 5 mM, 8 mM, 10 mM, 15 mM, 20 mM, 25 mM, 30 mM, 35 mM, 40 mM, 45 mM, 50 mM, or other such values within the range of about 1 mM to about 50 mM.
  • the buffer concentration within the formulation is from about 5 mM to about 15 mM, including about 5 mM, 6 mM, 7 mM, 8 mM, 9 mM, 10 mM, 1 1 mM, 12 mM, 13 mM, 14 mM, 15 mM, or other such values within the range of about 5 mM to about 1 mM.
  • the liquid pharmaceutical formulation comprises a therapeutically effective amount of the anti-CD40 antibody and succinate buffer or citrate buffer at a concentration that maintains the; pH of the formulation in the range of about pH 5.0 to about pH 7.0, preferably about pH 5.0 to about pH 6.5.
  • succinate buffer or "citrate buffer” is intended a buffer comprising a salt of succinic acid or a salt of citric acid, respectively.
  • the succinate or citrate counterion is the sodium cation, and thus the buffer is sodium succinate or sodium citrate, respectively.
  • any cation is expected to be effective.
  • Other possible succinate or citrate cations include, but are not limited to, potassium, ammonium, calcium, and magnesium.
  • the succinate or citrate buffer concentration within the formulation can be from about 1 mM to about 50 mM, including about 1 mM, 2 mM, 5 mM, 8 mM, 10 mM, 15 mM, 20 mM, 25 mM, 30 ⁇ mM, 35 mM, 40 mM, 45 mM, 50 mM, or other such values within the range of about 1 mM to about 50 mM.
  • the buffer concentration within the formulation is from about 5 mM to about 15 mM, including about 5 mM, 6 mM, 7 mM, 8 mM, 9 mM, 10 mM, 1 1 mM, 12 mM, 13 mM, 14 mM, or about 15 mM.
  • the liquid pharmaceutical formulation comprises the anti-CD40 antibody at a concentration of about 0.1 mg/ml to about 50.0 mg/ml, or about 5.0 mg/ml to about 25.0 mg ml, and succinate or citrate buffer, for example, sodium succinate or sodium citrate buffer, at a concentration of about 1 mM to about 20 mM, about 5 mM to about 15 mM, preferably about 10 mM.
  • succinate or citrate buffer for example, sodium succinate or sodium citrate buffer
  • the liquid pharmaceutical formulation comprising the anti-CD40 antibody and a buffer can further comprise an amount of an isotonizing agent sufficient to render the formulation near isotonic.
  • an isotonizing agent sufficient to render the formulation near isotonic.
  • the aqueous formulation has an osmolarity of about 240 mmol/kg to about 360 mmol/kg, preferably about 240 to about 340 mmol/kg, more preferably about 250 to about 330 mmol/kg, even more preferably about 260 to about 320 mmol/kg, still more preferably about 270 to about 310 mmol/kg.
  • the isotonizing agent can be any reagent capable of adjusting the osmotic pressure of the liquid pharmaceutical formulation of the present invention to a value nearly equal to that of a body fluid. It is desirable to use a physiologically acceptable isotonizing agent.
  • the liquid pharmaceutical formulation comprising a therapeutically effective amount of the anti-CD40 antibody and a buffer can further comprise components that can be used to provide isotonicity, for example, sodium chloride; amino acids such as alanine, valine, and glycine; sugars and sugar alcohols (polyols), including, but not limited to, glucose, dextrose, fructose, sucrose, maltose, mannitol, trehalose, glycerol, sorbitol, and xylitol; acetic acid, other organic acids or their salts, and relatively minor amounts of citrates or phosphates.
  • additional agents that are suitable for providing optimal tonicity of the liquid formulation.
  • the liquid pharmaceutical formulation comprising an anti-CD40 antibody and a buffer further comprises sodium chloride as the isotonizing agent.
  • concentration of sodium chloride in the formulation will depend upon the contribution of other components to tonicity. In some embodiments, the concentration of sodium chloride is about 50 mM to about 300 mM, about 50 mM to about 250 mM, about 50 mM to about 200 mM, about 50 mM to about 175 mM, about 50 mM to about 150 mM, about 75 mM to about 175 mM, about 75 mM to about 150 mM, about 100 mM to about 175 mM, about 100 mM to about 200 mM, about 100 mM to about 150 mM, about 125 mM to about 175 mM, about 125 mM to about 150 mM, about 130 mM to about 170 mM, about 130 mM to about 160 mM, about 135 mM to about 155
  • the concentration of sodium chloride is about 150 mM. In other such embodiments, the concentration of sodium chloride is about 150 mM, the buffer is sodium succinate or sodium citrate buffer at a concentration of about 5 mM to about 15 mM, the liquid pharmaceutical formulation comprises a therapeutically effective amount of the anti-CD40 antibody and the formulation has a pH of about pH 5.0 to about pH 7.0, about pH 5.0 to about pH 6.0, or about pH 5.5 to about pH 6.5.
  • the liquid pharmaceutical formulation comprises the anti-CD40 antibody at a concentration of about 0.1 mg/ml to about 50.0 mg/ml or about 5.0 mg ml to about 25.0 mg/ml, about 150 mM sodium chloride, and about 10 mM sodium succinate or sodium citrate, at a pH of about pH 5.5.
  • liquid pharmaceutical formulation of the present invention Protein degradation due to freeze thawing or mechanical shearing during processing of a liquid pharmaceutical formulation of the present invention can be inhibited by incorporation of surfactants into the formulation in order to lower the surface tension at the solution-air interface.
  • the pharmaceutical formulation comprises a therapeutically effective amount of the anti- CD40 antibody, a buffer, and further comprises a surfactant.
  • the liquid pharmaceutical formulation comprises an anti-CD40; antibody, a buffer, an isotonizing agent, and further comprises a surfactant.
  • Typical surfactants employed are nonionic surfactants, including polyoxyethylene sorbitol esters such as polysorbate 80 (Tween 80) and polysorbate 20 (Tween 20);
  • polyoxypropylene-polyoxyethylene esters such as Pluronic F68; polyoxyethylene alcohols such as Brij 35; simethicone; polyethylene glycol such as PEG400;
  • lysophosphatidyl choline lysophosphatidyl choline
  • polyoxyethylene-p-t-octylphenol such as Triton X-100.
  • Classic stabilization of pharmaceuticals by surfactants or eiiiulsifiers is described, for example, in Levine et al. (1991) J. Parenteral Sci. Technol. 45(3):160-165.
  • a preferred surfactant employed in the practice of the present invention is polysorbate 80.
  • a surfactant is included, it is typically added in an amount from about 0.001 % to about 1.0% (w/v), about 0.001% to about 0.5%, about 0.001% to about 0.4%, about 0.001% to about 0.3%, about 0.001 % to about 0.2%, about 0.005% to about 0.5%, about 0.005% to about 0.2%, about 0.01% to about 0.5%, about 0.01% to about 0.2%, about 0.03% to about 0.5%, about 0.03% to about 0.3%, about 0.05% to about 0.5%, or about 0.05% to about 0.2%.
  • the liquid pharmaceutical formulation comprises a therapeutically effective amount of the anti-CD40 antibody
  • the buffer is sodium succinate or sodium citrate buffer at a concentration of about 1 mM to about 50 mM, about 5 mM to about 25 mM, or about 5 mM to about 15 mM
  • the formulation has a pH of about pH 5.0 to about pH 7.0, about pH 5.0 to about pH 6.0, or about pH 5.5 to about pH 6.5
  • the formulation further comprises a surfactant, for example, polysorbate 80, in an amount from about 0.001% to about 1.0% or about 0.001 % to about 0.5%.
  • Such formulations can optionally comprise an isotonizing agent, such as sodium chloride at a concentration of about 50 mM to about 300 mM, about 50 mM to about 200 mM, or about 50 mM to about 150 mM.
  • the liquid pharmaceutical formulation comprises the anti-CD40 antibody at a concentration of about 0.1 mg/ml to about 50.0 mg/ml or about 5.0 mg/ml to about 25.0 mg/ml, including about 20.0 mg/ml; about 50 mM to about 200 mM sodium chloride, including about 150 mM sodium chloride; sodium succinate or sodium citrate at about 5 mM to about 20 mM, including about 10 mM sodium succinate or sodium citrate; sodium chloride at a concentration of about 50 mM to about 200 mM, including about 150 mM; and optionally a surfactant, for example, polysorbate 80, in an amount from about 0.001% to about 1.0%, including about 0.001% to about 0.5%; where the liquid pharmaceutical formulation
  • the liquid pharmaceutical formulation can be essentially free of any preservatives and other carriers, excipients, or stabilizers noted herein above.
  • the formulation can include one or more preservatives, for example, antibacterial agents, pharmaceutically acceptable carriers, excipients, or stabilizers described herein above provided they do not adversely affect the physicochemical stability of the anti-CD40 antibody.
  • acceptable carriers, excipients, and stabilizers include, but are not limited to, additional buffering agents, co-solvents, surfactants, antioxidants including ascorbic acid and methionine, chelating agents such as EDTA, metal complexes (for example, Zn-protein complexes), and biodegradable polymers such as polyesters.
  • Carriers as used herein include pharmaceutically acceptable carriers, excipients, or stabilizers that are nontoxic to the cell or mammal being exposed thereto at the dosages and concentrations employed. Often the physiologically acceptable carrier is an aqueous pH buffered solution.
  • physiologically acceptable carriers include buffers such as phosphate, citrate, succinate, and other organic acids; antioxidants including ascorbic acid; low molecular weight (less than about 10 residues) polypeptides; proteins, such as serum albumin, gelatin, or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, arginine or lysine; monosaccharides, disaccharides, and other carbohydrates including glucose, mannose, or dextrins; chelating agents such as EDTA; sugar alcohols such as mannitol or sorbitol; salt-forming counterions such as sodium; and/or nonionic surfactants such as TWEEN, polyethylene glycol (PEG), and Pluronics.
  • buffers such as phosphate, citrate, succinate, and other organic acids
  • antioxidants including ascorbic acid
  • low molecular weight (less than about 10 residues) polypeptides proteins
  • liquid pharmaceutical formulation or other pharmaceutical composition described herein After the liquid pharmaceutical formulation or other pharmaceutical composition described herein is prepared, it can be lyophilized to prevent degradation. Methods for lyophilizing liquid compositions are known to those of ordinary skill in the art. Just prior to use, the composition may be reconstituted with a sterile diluent (Ringer's solution, distilled water, or sterile saline, for example) that may include additional ingredients. Upon reconstitution, the composition is preferably administered to subjects using those methods that are known to those skilled in the art.
  • a sterile diluent Finger's solution, distilled water, or sterile saline, for example
  • the anti-CD40 antibody-containing pharmaceutical: composition may be a composition as described in co-owned International Patent Application No.
  • a pharmaceutical composition for use in the combination therapy of the invention may comprise (i) an anti- CD40 antibody, a buffering agent to maintain the pH of the composition between around pH 5.0 and pH 7.0, and (iii) an amount of arginine-HCl sufficient to render the liquid composition near isotonic.
  • the buffering agent may be a citrate/citric acid buffer.
  • the composition may further comprise a non-ionic surfactant and/or L-methionine as further stabilizing agents.
  • the composition may have an osmolarity of about 240 mmol/kg to about 360 mmol/kg.
  • the concentration of the buffering agent may be from about 5 mM to about 100 mM, from about 5 mM to about 20 mM, or from about 5 mM to about 15 mM (e.g., 10 mM).
  • the composition may have a pH of from pH 5.0 to pH 6.0 (e.g., around pH 5.5).
  • the composition may comprise arginine-HCl at a concentration of about 50 mM to about 200 mM, or from about 100 mM to about 175 mM (e.g., about 150 mM).
  • the composition may further comprise the surfactant polysorbate, for example at a concentration of about 0.001% to about 1.0% (w/v), or at a concentration of about 0.025% to about 0.1% (w/v).
  • the composition may further comprise methionine at a concentration of about 0.5 mM to about 20.0 mM or at a concentration of about 1.0 mM to about 20.0 mM (e.g., about 5.0 mM).
  • the anti-CD40 antibody may be present in the composition at about 0.1 mg ml to about 50.0 mg/ml, or at about 1.0 mg/ml to about 35.0 mg/ml, or at about 10.0 mg/ml to about 35.0 mg/ml.
  • Any pharmaceutical composition comprising bendamustine as the therapeutically active component can be used in the methods of the invention. These will contain bendamustine and a pharmaceutically acceptable carrier or excipient, e.g., a
  • Suitable pharmaceutical compositions are well known in the art.
  • therapeutically active component is intended that the relevant therapeutic agent is specifically incorporated into the composition to bring about a desired therapeutic response with regard to treatment of a disease or condition within a subject when the pharmaceutical composition is administered to that subject.
  • the bendamustine component is administered at concentrations that are "therapeutically effective" to treat a disease or condition associated with neoplastic B-cell growth.
  • the bendamustine may be administered by any appropriate route of
  • Bendamustine is normally administered intravenously. Methods to accomplish this administration are known to those of ordinary skill in the art.
  • Bendamusitine is normally administered in cycles of treatment, each cycle comprising administration of bendamustine at about 60 mg/m 2 to about 120 mg/m 2 on day 1 and day 2 of a treatment cycle, preferably at about 90 mg/m 2 on day 1 and day 2 of a treatment cycle.
  • the cycle is generally repeated every three weeks (21 days) or every four weeks (28 days).
  • a usual course of treatment consists of four to eight cycles in total, including, for example, four cycles, five cycles, six cycles, seven cycles, or eight cycles.
  • the bendamustine may be used at 60-120 mg/m 2 , 60-1 10 mg/m2, 60-100 mg/m2, 60-90 mg/m2, 70-120 mg/m2, 70-1 10 mg/m2, 70-100 mg/m2, 70-90 mg/m2, 80-120 mg/m2, 80-1 10 mg/m2, 80-100 mg/m2, or 80-95 mg/m2 (e.g., at 90 mg/m 2 ).
  • the skilled person will readily be able to select an appropriate bendamustine regimen for use in the combination therapy of the invention when equipped with the disclosure provided herein.
  • the bendamustine regimen will preferably be repeated every four weeks, but may be repeated every three weeks, every four weeks, every five weeks, every six weeks, every seven weeks, or every eight weeks, if desired.
  • the combination therapy of the invention may enable lower doses of bendamustine to be used while retaining therapeutic efficacy, thereby allowing the bendamustine regimen to be repeated more frequently, such as every week or every two weeks.
  • the bendamustine can be administered for any desired number of cycles, e.g., 1- 20 cycles, preferably 3-15 cyles, more preferably 5-10 cycles.
  • composition “comprising” encompasses “including” as well as “consisting”.
  • a composition “comprising” X may consist exclusively of X or may include something additional, e.g., X + Y.
  • the anti-CD40 antibody used in the examples below is the monoclonal antibody HCD122 (formerly known as CHIR-12.12).
  • HCD122 previously known as CHIR-12.12
  • HCD122 is a potent CD40 antagonist that blocks CD40L-induced proliferation and survival. HCD122 prevents binding of CD40L to cell surface CD40 and is able to compete with unbound, soluble CD40L. HCD122 does not internalize upon binding cell surface, human CD40.
  • HCD122 When HCD122 was evaluated for its ability to induce antibody- dependent cell-mediated cytotoxicity (ADCC), the potency and maximal amount of cell lysis induced by HCD122 was greater than that observed for the anti-CD20 antibody, rituximab, in both primary NHL tumor cells and cell lines (Daudi, Namalwa). Thus, HCD122 is anticipated to have activity against lymphoid malignancies by blocking the CD40/CD40L activation pathway and via ADCC.
  • Example 1 Determination of MTD for Bendamustine in SCID Mice
  • This experiment was designed to establish the maximum tolerated dose (MTD) for bendamustine in SCID mice.
  • Two regimens were tested: once daily for two days (which is the clinical dose regimen) and once daily for 5 days to provide a more sustained dose regimen.
  • Each animal was monitored for weight loss over the 14- day study period during the active dosing phase.
  • NTRm Number of non-treatment-related deaths due to metastatsis.
  • the Group 3 and Group 4 dosing regimens identified in Example 1 are tested for efficacy in the human xenograft models RL and SU-DHL-4 in SCID mice.
  • the RL cell line (ATCC; CRL-2261) is a human B cell lymphoma cell line established from a 52 year old Caucasian male patient with NHL.
  • the SU-DHL-4 cell line (DSMZ; ACC , 495) is a human B cell lymphoma cell line established frorri the peritoneal effusion of a 38 year old man with B-NHL (diffuse large cell, cleaved cell type). These cells lines are both reported to be negative for the Epstein-Barr virus genome, in contrast to many of the common lymphoma cell lines used in the field. The use of cell lines that are positive for
  • Example 3 Combination Study of HCD122 Plus Bendamustine Human DLBCL Xenograft Models.
  • the human monoclonal antibody HCD122 and bendamustine have each shown anti-tumor efficacy in RL and SU-DHL-4 lymphoma models when used alone.
  • HCD122 The anti-tumor activity of HCD122 is tested in RL DLBCL xenograft models in combination with bendamustine in CB17.SCID mice.
  • HCD 22 is administered at high (30 mg/kg) and low (10 mg/kg) dose levels in combination with the fully efficacious dose of bendamustine (30 . mg kg, dosed once daily for two days), and compared to comparable dosing regimens for these single agents, to study HCD122 effects on tumor growth delay.
  • This study design is thought to best represent the clinical setting where bendamustine is given at the established efficacious dose level.
  • HCD122 and the negative control human IgGl antibody, are each administered by intraperitoneal injection (ip) on a once-a-week schedule, or bi-weekly (once every two weeks) and the length of treatment is 4 weeks, where the dose of HCD122 is 10 mg/kg or 30 mg/kg.
  • HCD122 is administered alone (as a single agent) or in combination with bendamustine.
  • the bendamustine is administered intravenously (iv), alone or in combination with HCD122 ip administration, at the efficacious dose of 30 mg/kg, once daily on day 1 and day 2 of the treatment period.
  • iv intravenously
  • HCD122 ip administration at the efficacious dose of 30 mg/kg, once daily on day 1 and day 2 of the treatment period.
  • length then width are measured with a digital caliper.
  • the animals' weights are recorded and evaluated as a per group average.
  • TGI tumor growth inhibition
  • HCD122 is combined with a sub-efficacious dose of bendamustine to reveal potential combination effects on tumor growth during the dosing period.
  • Example 4 Phase lb Study of HCD122 Administered Intravenously in Combination with Bendamustine in Patients with CD40+ Follicular Lymphoma Who Are Refractory to Rituximab Follicular lymphoma (FL) is the most common indolent non-Hodgkin's lymphoma (NHL) and the second most common form of NHL worldwide, comprising approximately 20% to 25% of all NHL cases in the United States and Europe (Salles (2007) "Clinical featuers, prognosis and treatment of follicular lymphoma," Hematology Am. Soc. Hemtol. Educ. Program 216-25. The incidence of FL is increasing, with over 24,000 new cases diagnosed worldwide each year (Friedberg (2008) Semin.
  • Hematol. 45(Suppl. 2):S2-S6) The majority of patients diagnosed with FL are 50 years and older, present with widespread disease at diagnosis, and have a median survival of 8 to 15 years from initial diagnosis (Peterson et al. (2003) J. Clin. Oncol. 21(1):5-15; Swenson et al. (2005) J. Clin. Oncol. 23(22):5019-5026; Liu et al. (2006) J. Clin. Oncol.
  • Therapy options for symptomatic patients after initial diagnosis include chemotherapy, rituximab monotherapy, rituximab and chemotherapy, or
  • radioimmunotherapy National Comprehensive Cancer Network (NCCN), v 1.2010, NCCN clinical practice guidelines in oncology, NCCN, Fort Washington, PA).
  • NCN National Comprehensive Cancer Network
  • doxorubicin doxorubicin
  • vincristine doxorubicin
  • prednisone CHOP
  • cyclophosphamide cyclophosphamide, vincristine, and prednisone (CVP); and fludarabine.
  • Radioimmunotherapy with yttrium-90- labeled ibritumomab tiuxetan or iodine- 131- labeled tositumomab has also been used in this setting.
  • second-line treatment options include rituximab plus chemotherapy (e.g. R-CHOP, R-CVP), fludarabine-based chemotherapy, bendamustine, or high-dose chemotherapy with autologous or allogeneic stem-cell rescue.
  • the anti-CD20 monoclonal antibody, rituximab has been a valuable addition to the treatment of B-cell NHL and has improved response rates in FL.
  • 50% of patients with relapsed or refractory CD20-positive FL do not respond to initial therapy with rituximab, and approximately 60% of patients who were previously treated with rituximab no longer benefit from retreatment (Tay et al. (2010) Blood Rev. 24(2):69- 82).
  • the alkylating agent, bendamustine was recently approved as a single agent for rituximab-refractory NHL, and is an effective treatment option for indolent B-cell NHLs that are refractory to rituximab (Friedberg et al. (2008) J. Clin. Oncol. 26:204-210; Kahl et al. (2010) Cancer 116(1): 106-114).
  • preliminary studies with bendamustine plus rituximab demonstrate improved progression free survival (PFS) and complete response (CR) rates compared with R-CHOP (Rummel et al. (2009) Bloodd 114:168-169 (Abstract 405).
  • NHL has resulted in improved CR rates and survival rates in some settings, the lack of any significant improvement in overall survival indicates a continued need for novel drugs and interventions (Tay et al. (2010) Blood Rev. 24(2):69 82). Also, few treatment options for FL patients who are refractory to rituximabcontaining regimens are available. Those that are available, such as radioimmunotherapies, have not been incorporated into broad clinical use. In particular, an unmet medical need exists for effective treatments for patients who are refractory to rituximab and have received at least one prior chemotherapeutic regimen.
  • HCD122 to bendamustine therapy will result in a greater response rate with a more durable response in this population of patients than when either agent is used alone.
  • This phase lb, multicenter, open-label study will investigate the safety, pharmacokinetics, pharmacodynamics, and preliminary efficacy of HCD122 in combination with bendamustine in patients with documented CD40 + follicular lymphoma (FL) that is refractory to treatment with rituxmab and who have received treatment with at least one prior chemotherpauetic regimen.
  • FL follicular lymphoma
  • the patient population is adult patients ( 18 years of age or older) having this clinical presentation.
  • "refractory” is defined as disease progression during rituximab treatment or disease progression within 6 months of the last rituximab dose.
  • DLTs that primarily contribute to the determination of the MTD are identified during cycle 1 of dose escalation.
  • a DLT is defined as an adverse event (AE) or abnormal laboratory value assessed as clinically relevant and related to study treatment.
  • AE adverse event
  • Type, frequency, and severity of AEs are monitored during the dose escalation and dose expansion phases, as are changes in hematology and blood chemistry valiues, assessments of physical examinatons, vital signs, and
  • the study is subdivided into four sequential periods, including screening (where patients having documented CD40 + follicular lymphoma are identified and enrolled), combination treatment, HCD122 maintenance treatment, and follow-up.
  • the combination treatment period begins on day 1 of cycle 1. During this period, HCD122 is administered in combination with bendamustine for 6 treatment cycles or until disease progression or study discontinuation. Continuation of the combination treatment period for up to 8 cycles may be considered. Treatment cycles are 28-days. During the 28-day cycles of the combination treaunent period, both HCD122 and benadmustine are administered intravenously (iv). During cycle 1 of dose escalation, 90 mg/m 2 bendamustine is administered over 60 minutes on days 1 and 2; HCD122 is administered on days 3 and 5 at the assigned dose (estimated duration of HCD122 infusion is 2-6 hours).
  • HCD122 is administered on days 1 and 15 at the assigned dose (dose escalation phase) or at the MTD (dose expansion phase).
  • HCD122 is administered before bendamustine.
  • the HCD122 maintenance treatment period follows the combination treatment period for patients enrolled in the dose escalation and dose expansion phases of the trial. During this period, HCD122 is administered as a single agent biweekly (i.e., once every 14 days) on day 1 and day 15 of the maintenance treatment period, at the assigned dose (dose escalation phase) or MTD (dose expansion phase) until disease progression or study discontinuation. A patient may be considered to begin the HCD122 maintenance treatment period prior to the completion of the sixth combination treatment cycle if the patient is unable to tolerate additional bendamustine infusions and remains eligible for the study.
  • the end-of-treatment (EOT) visit occurs within seven days after the last administration of either HCD122 or bendamustine. All participating patients must complete this visit even if they have had to discontinue prematurely.
  • the follow-up period starts after the EOT visit and continues until the completion of all follow-up assessments.
  • the visits and assessments that occur during the follow-up period may include assessment of AEs 28 days following the last dose of study treatment; and tumor response assessments until disease progression or start of new anti-cancer therapy (every 3 months).
  • tgc atg caa get cga caa act cca ttc act ttc ggc cct ggg acc aaa 384 Cys Met Gin Ala Arg Gin Thr Pro Phe Thr Phe Gly Pro Gly Thr Lys 115 120 125
  • Glu Trp Val Ala Val lie Ser Tyr Glu Glu Ser Asn Arg Tyr His Ala 65 70 75 80
  • agt gac tgc aca gag ttc act gaa acg gaa tgc ctt cct tgc ggt gaa Ser Asp Cys Thr Glu Phe Thr Glu Thr Glu Cys Leu Pro Cys Gly Glu
  • ccc tgc cca gtc ggc ttc ttc tec aat gtg tea tct get ttc gaa aaa 480 Pro Cys Pro Val Gly Phe Phe Ser Asn Val Ser Ser Ala Phe Glu Lys 145 150 155 160 tgt cac cct tgg aca agg tec cca gga teg get gag age cct ggt ggt 528 Cys His Pro Trp Thr Arg Ser Pro Gly Ser Ala Glu Ser Pro Gly Gly
  • agt gac tgc aca gag ttc act gaa acg gaa tgc ctt cct tgc ggt gaa 192 Ser Asp Cys Thr Glu Phe Thr Glu Thr Glu Cys Leu Pro Cys Gly Glu
  • Lys Thr lie Ser Lys Ala Lys Gly Gin Pro Arg Glu Pro Gin Val Tyr
  • Ala Arg Asp Gly Gly lie Ala Ala Pro Gly Pro Asp Tyr Trp Gly Gin
  • Lys Thr lie Ser Lys Ala Lys Gly Gin Pro Arg Glu Pro Gin Val Tyr

Abstract

La présente invention concerne de nouvelles utilisations d'anticorps anti-CD40 en combinaison avec de la bendamustine dans le traitement de maladies ou d'états associés à la croissance des cellules B néoplasiques. L'invention est particulièrement utile pour le traitement de patients auxquels on a précédemment administré (i) l'anticorps monoclonal anti-CD20 chimère rituximab ou (iii) une thérapie combinée comportant du rituximab et un régime chimiothérapeutique.
PCT/US2011/062584 2010-11-30 2011-11-30 Utilisation d'anticorps anti-cd40 en thérapie combinée contre des cancers associés aux cellules b WO2012075111A1 (fr)

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